Nir-fluorescent cyanine dyes, their synthesis and biological use

ABSTRACT

The invention includes new water-soluble NIR fluorochromes, e.g., for biomedical imaging. The new dyes are highly stable, asymmetric cyanine compounds, characterized by 1) superior chemical stability, 2) excellent optical properties (e.g., high quantum yield), 3) bio-compatibility, 4) conjugatability and 5) ideal in vivo imaging properties. Monoactivated hydroxysuccinimide esters of the new dyes are highly reactive with peptides, metabolites, proteins, peptide-folate conjugates, and other biological macromolecules and affinity ligands, forming stable complexes. Affinity molecules tagged with the new dyes can be used, for example, for imaging of tumors in vivo.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/368,962, filed on Mar. 29, 2002. Thecontents of this application is hereby incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

This invention relates to chromophores for optical imaging, and moreparticularly to asymmetric near infrared (NIR) chromophores and methodsfor their synthesis and use.

BACKGROUND OF THE INVENTION

Light-based imaging methods provide a non-invasive avenue for extractingbiological information from living subjects. These methods measurevarious native parameters of tissues through which photons can travel.Such parameters include absorption, scattering, polarization, spectralcharacteristics, and fluorescence. While light in the visible range(i.e., 400-650 nm) can be used for analysis of tissue surface structuresand intravital microscopy of relatively shallow tissues (i.e., less thanabout 800 μm below the tissue surface), imaging of deeper tissuesgenerally requires the use of near infrared (NIR) light. NIR radiation(approx. 600-1000 nm) exhibits tissue penetration of up to tencentimeters, and can accordingly be used for imaging internal tissues(see, e.g., Wyatt, Phil. Trans. R. Soc. London B, 352:701-706, 1997; andTromberg et al., Phil. Trans. R. Soc. London B. 352:661-667, 1997).

Besides being non-invasive, NIR fluorescence imaging methods offer anumber of advantages over other imaging methods: they provide generallyhigh sensitivity, do not require exposure of test subjects or labpersonnel to ionizing radiation, can allow for simultaneous use ofmultiple, distinguishable probes (important in molecular imaging), andoffer high temporal and spatial resolution (important in functionalimaging and in vivo microscopy, respectively).

In NIR fluorescence imaging, filtered light or a laser with a definedbandwidth is used as a source of excitation light. The excitation lighttravels through body tissues. When it encounters an NIR fluorescentmolecule (i.e., a “contrast agent”), the excitation light is absorbed.The fluorescent molecule then emits light that has, for example,detectably different spectral properties (e.g., a slightly longerwavelength) from the excitation light. Despite good penetration ofbiological tissues by NIR light, conventional NIR fluorescence probesare subject to many of the same limitations encountered with othercontrast agents, including low target/background ratios.

A number of reflectance and tomographic imaging systems have recentlybeen developed to detect NIR fluorescence in deep tissues, including inpatients (Ntziachristos et al., Proc. Natl. Acad. Sci. USA., 97:2767-72(2000)). Nonetheless, there is a need for a new generation ofbiocompatible fluorochromes. Indocyanine green (ICG) has been usedclinically for over 20 years with few side effects (Hope-Ross et al.,Ophthalmology, 101:529-533 (1994)), but its use in designing targetedagents is limited by the fact that monoderivatized activated precursorsare not available. Moreover, ICG is hydrophobic and exhibits a highdegree of albumin binding and nonlinear fluorescence. Syntheticfluorochromes have been plagued by problems such as significant spectralbroadening as wavelengths increase, low quantum yield, photoinstability,chemical instability with increasing red-shift, and a tendency toaggregate as a result of large planar surfaces and/or hydrophobicity.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery and synthesis of newwater-soluble NIR chromophores for biomedical imaging. The newchromophores are highly stable, asymmetric cyanine compounds,characterized by 1) superior chemical stability, 2) excellent opticalproperties (e.g., high quantum yield), 3) bio-compatibility, 4)conjugatability, and 5) ideal in vivo imaging properties. Monoactivatedhydroxysuccinimide esters of the new chromophores are highly reactivewith peptides, metabolites, proteins, peptide-folate conjugates, andother biological macromolecules and affinity ligands, forming stablecomplexes that can be used as biocompatible probes. Affinity moleculestagged with the new chromophores can be used, for example, for imagingof tumors in vivo.

In one aspect, the invention features asymmetrical chromophores havingthe following formula:

L is a conjugated linker moiety (e.g., L can be (CH═CH—)_(n)CH, wheren=1,2, 3, or 4, or can include one or more conjugated ring structures).

R-₁₋₁₂ can be, independently, hydrogen, substituted or unsubstitutedalkyl groups (where substituted means that one or more hydrogen atomsare replaced by carbon-, nitrogen-, oxygen-, phosphorus-, and/orhydrogen-containing functional groups), substituted or unsubstitutedalkenyl groups, substituted or unsubstituted alkynyl groups, substitutedor unsubstituted aryl groups, sulfur-containing functional groups,phosphorus-containing functional groups, oxygen-containing functionalgroups, or nitrogen-containing functional groups; and

X and Y can be the same or different, and can be, for example, —O—, —S—,—NH— (or a substituted variant thereof where H is replaced by an alkyl,alkenyl, alkynyl, aryl, or other moiety), or substituted orunsubstituted methylene (—CH₂). Thus, for example, X and Y can both bedimethylmethylene groups (i.e., —C(CH₃)₂—).

One or more of R₁₋₁₂ can include a reactive group for conjugation to amacromolecule (e.g., an amino group for conjugation with an carboxylatederivative, or vice versa) to form a molecular probe (e.g., an imagingprobe). In some cases, one or more of R-₁₋₁₂ can include at least onesulfate, sulfonate, phosphate, phosphonate, halide, nitro, nitrile,nitrate, or carboxylate group. In particular embodiments, for example,R₁, R₃, R₅, R₆, R₉, R₁₀, and R₁₂ can all be hydrogen, R₂ and R₄ can bothbe —₃ ⁻, R₇ can be —CH₂CH₃, R₈ can be (CH₂)₄SO₃ ⁻, R₁₁ can be CO₂H, andX and Y can be —C(CH₃)₂—.

In another aspect, the invention features asymmetrical chromophoreshaving the formula:

where L, R₇, R₈, X, and Y are defined as above; R₁₃ is C(O)OR₁₄ orNHC(O)CH₂J; R₁₄ is H or

Z is a group of nonmetallic atoms necessary for forming a substituted orunsubstituted, condensed aromatic ring or ring system. Thus, forexample, Z can be either of:

where R₂₋₆ are defined as above.

In the case where Z is

R₁, R₃, R₅, and R₆ can be hydrogen, R2 and R4 can be —SO₃ ⁻, can be—CH₂CH₃, R₈ can be (CH₂)₄SO₃ ⁻, and X and Y can be —C(CH₃)₂—.

In the case where Z is

R₂, R₅, and R₆ can be hydrogen, R₃ can be —SO₃ ⁻, R₇ can be —CH₂CH₃, R₈can be (CH₂)₄SO₃ ⁻, and X and Y can be —C(CH₃)₂.

In a further aspect, the invention features asymmetrical chromophoreshaving the formula:

where X is selected from the group consisting of:

R₈, R₁₃ and R₁₄ are defined as above and n=2 or 3.

Embodiments can include one or more of the following.

R₁₄ can be H

J can be Cl or I. R₈ can be CH₃ or (CH₂)₄SO₃ ⁻.

The invention also features molecular and/or imaging probes that includethe new chromophores.

In another aspect, the invention features methods of gene sequencerecognition using fluorescently labeled nucleic acid recognitionmolecules, including DNA, RNA, modified nucleic acid, PNA molecularbeacon, or other nucleic acid binding molecules. The methods include theuse of one or more of the chromophores described above, together withany one or combination of well-known techniques such as hybridization,ligation, cleavage, recombination, synthesis, sequencing, mutationdetection, real-time polymerase chain reactions, in situ hybridization,and the use of microarrays.

The invention also features in vivo imaging methods (e.g., NIR imagingin a human or animal) for imaging tissue (e.g., a living tissue, e.g., adiseased tissue). The methods include a) conjugating to a targetingligand (e.g., an antibody, a protein, a peptide, a receptor bindingligand, a small ligand, or a carbohydrate) a chromophore as describedabove; b) combining the conjugated chromophore with a suitable excipientto form an injectable or otherwise administerable formulation; c)administering the formulation to a tissue; and d) detecting theconjugated chromophore (e.g., by using NIR spectroscopy) in the tissueto provide a fluorescence image of the tissue. The imaging method can beused, for example, in the detection of disease (e.g., cancer, CNSdiseases, cardovascular diseases, arthritis) at an early stage or at themolecular level; for characterization of disease sensitivity, prognosis,and/or molecular profile; or for determination of drug efficacy at themolecular level, or response to particular drugs, to optimize drugdosage in individual patients, or for drug discovery in vivo. The samemethods can be used for in vitro imaging, although, in that case, thecombining with an excipient and administering steps can generally beomitted.

The invention also features in vivo enzyme sensing methods. The methodsinclude a) conjugating, to an enzyme-activatable molecule, a chromophoreas described above; b) combining the conjugated chromophore with asuitable excipient to form an injectable formulation; c) injecting theformulation into a tissue (e.g., so that the injected chromophore willinteract with specific enzymes and cause optical signal changes); and d)detecting the conjugated chromophore in the tissue to provideinformation about targeted enzymes. As above, the same method can beused for in vitro enzyme sensing (e.g., without the combination andinjection steps).

The chromophores described above can also be used as free dyes for invivo imaging.

The fluorescence signal generated by the chromophores described above,or conjugates thereof, whether collected by tomographic, reflectance,endoscopic, video imaging technologies, or other methods, and whetherused quantitatively or qualitatively, is also considered to be an aspectof the invention.

As used herein, the terms “fluorochrome” and “fluorochrome dye” bothrefer to chromophores that are able to absorb energy at a ground stateand emit fluorescence light from an excited state. The chromophores canbe conjugated with other molecules (e.g., biological macromolecules) toform molecular probes (e.g., imaging probes, e.g., NIR fluorescenceprobes).

As used herein, the term “asymmetrical chromophore” refers to achromophore of formula A-L-B, where A and B are non-identicalunsaturated moieties, and L is a linker that includes conjugate doublebonds.

The invention provides several advantages. For example, the newchromophores offer: 1) peak fluorescence in or close to the 700-900 nmrange, which is ideal for optical in vivo imaging, 2) high quantumyield, 3) narrow excitation/emission spectra, 4) high chemical- andphoto-stability, 5) low or no toxicity, 6) water-solubility, 7)biocompatibility, biodegradability, and excretability, 8) availabilityof monofunctional derivatives as a platform technology, and 9)commercial viability and scalability of production for large quantitiesrequired for human use. Moreover, the new chromophores are asymmetric toavoid stacking of large planar surfaces, contain multiple hydrophilicgroups, and can be prepared as monohydroxy succinimide esters forbinding to biomolecules such as peptides, metabolites, proteins,targeting ligands, DNA and other biomolecules.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic representation of the synthesis of two NIRchromophores of the invention, referred to herein as NIR1 and NIR2.

FIG. 2 is a schematic representation of the synthesis of two NIRchromophores of the invention, referred to herein as NIR3 and NIR4.

FIG. 3A is a schematic representation of the synthesis of four NIRchromophores of the invention, referred to herein as NIR5, NIR6, NIR7,and NIR8

FIG. 3B is a schematic representation of the synthesis of syntheticintermediates 8 and 9, used in the synthesis of NIR7 and NIR8.

FIGS. 4A and 4B are a pair of spectra corresponding to the absorptionspectra of NIR1, NIR2, NIR3, and NIR4 (4A) and the fluorescence(excitation and emission) spectra of NIR1 and NIR2 (4B).

FIG. 5A is a schematic representation of the activation of NIR2 withN-hydroxysuccinimide.

FIG. 5B is a set of high performance liquid chromatography (HPLC) tracesof NIR2 before (top) and after (bottom) activation.

FIG. 5C is a schematic representation of the conversion of NIR5, NIR6,NIR7, and NIR8 to NIR9, NIR10, NIR11, and NIR12.

FIG. 5D is a set of high performance liquid chromatography (HPLC) tracesof of NIR10 and the NIR10-peptide conjugate.

FIG. 5E is a spectrum corresponding to the fluorescence (excitation andemission) spectra of NIR10-peptide conjugate.

FIG. 6 is a digitized photograph showing the fluorescences of NIR1 (well1), NIR2 (well 2), NIR3 (well 3), NIR4 (well 4), and indocyanine green(IGC; well 5) in response to white light (“light”) and two NIR frequencyranges (i.e., “700 nm” and “800 nm”).

FIG. 7 is a bar graph of the fluorescence intensity (y-axis) of NIR2attached to a PEGylated graft copolymer having a lysine backbone (i.e.,an “NIR2/PGC Probe”) before (white bars) and after (black bars) cleavageby trypsin for 3 hours. The numbers on the x-axis represent the numberof NIR2 residues per PCG molecule.

FIG. 8 is a schematic representation of the coupling of a folate-peptideconjugate to NIR2.

FIG. 9 is a digitized photograph of a tumor-bearing mouse, imaged usingfluorescence imaging 4 hours after injecting the mouse withfolate-derivatized NIR2.

FIG. 10 is a graphical representation corresponding to the cellularuptake of ³H-folate in the KB and HT1080 tumor cell lines.

FIG. 11 is a digitized photograph of KB and HT1080 tumor cells incubatedwith NIR2-folate probe (0.1 μm) for 30 minutes at 37° C.

FIGS. 12A, 12B, 12C, and 2D are digitized photographs of FR expressionand hematoxylin-eosin staining of KB and HT1080 tumors.

FIG. 13A is a digitized photograph of a white light image obtained 24hours after intravenous injection of the NIR2-folate probe in arepresentative animal.

FIG. 13B is a digitized photograph of of enlarged NIRF images of thechest tumors.

FIG. 13C is a digitized photograph of of enlarged NIRF images of the lowabdomen KB tumors.

FIG. 13D is a digitized photograph of a white light image obtained 24hours after intravenous injection of the NIR2-folate probe in arepresentative animal.

FIG. 14 is a graphical representation of the in vivo fluorescence signalof tumors and normal tissues.

FIG. 15 is a graphical representation of time course of KB tumor withvarious probes.

Unless otherwise noted, like reference symbols in the various drawingsindicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to highly stable, water-soluble, asymmetriccyanine compounds and their use as chromophores. In general, the newcompounds include at least one reactive functional group (e.g., amono-reactive carboxyl group) that can be used for labeling (i.e., achromophore attachment moiety). When multiple chromophores are attachedto a single macromolecule, fluorescence quenching can be observed. Thenew biocompatible chromophores, and molecular probes made therefrom,incorporate these properties, and can be used for in vivo detection ofspecific protease activity, particularly for those proteases that playkey roles in different aspects of cancer growth, metastases formation,and angiogenesis (Weissleder et al., Nature Biotech, 17:375-378, 1999;Tung et al., Canc. Res., 2000:4953-4958,2000; Bremer et al., Nat. Med.,7:743-748,2001). The chromophores can, for example, be attached to apartially PEGylated graft copolymer (PGC) with a polylysine backbone(Bogdanov et al., Adv. Drug Deliv. Rev., 16:335-348, 1995). The probesgenerally have minimum fluorescence signal in their native states andbecome highly fluorescent after enzyme-mediated release offluorochromes, resulting in signal amplification. Besides being usefulfor imaging, the new dyes can be used in a large range ofbiotechnological applications, such as DNA sequencing, molecular beaconsand protease assays.

The chromophore attachment moiety can be any biocompatible backbone thatallows one or a plurality of chromophores to be covalently linkedthereto. In one embodiment, the chromophore attachment moiety is apolymer, for example, a polypeptide, a polysaccharide, a nucleic acid,or a synthetic polymer. Alternatively, the chromophore attachment moietyis a monomeric, dimeric, or oligomeric molecule. Polypeptides useful asthe chromophore attachment moiety include, for example, polylysine,albumins, and antibodies. Poly(L-lysine) is a useful polypeptidechromophore attachment moiety. Other useful chromophore attachmentmoieties include synthetic polymers such as polyglycolic acid,polylactic acid, polyglutamic acid, poly(glycolic-co-lactic) acid,polydioxanone, polyvalerolactone, poly-ε-caprolactone,poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), polytartronic acid,and poly(β-malonic acid).

Activation sites can be located in the chromophore attachment moiety,e.g., when the chromophores are linked directly to ε-amino groups ofpolylysine. Alternatively, each chromophore can be linked to thechromophore attachment moiety by a spacer, e.g., a spacer containing achromophore activation site. The spacers can be oligopeptides.Oligopeptide sequences useful as spacers (or in spacers) include:Arg-Arg; Arg-Arg-Gly; Gly-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:1);His-Ser-Ser-Lys-Leu-Gln-Gly (SEQ ID NO:2);Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys(FITC)-Gly-Asp-Glu-Val-Asp-Gly-Cys(QSY7)-NH₂(SEQ ID NO:3); RRK(FITC)C-NH₂ (SEQ ID NO: 4); GRRK(FITC)C-NH₂ (SEQ IDNO:5); GRRRRK(FITC)C-NH₂ (SEQ ID NO:6); GRRGRRK(FITC)C-NH₂ (SEQ IDNO:7); GFGSVQ:FAGK(FITC)C-NH₂ (SEQ ID NO:8); GFLGGK(FITC)C-NH₂ (SEQ IDNO:9); Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys(FITC)-Cys-NH₂ (SEQ ID NO: 10);Gly-D-Phe-Pip-Arg-Ser-Gly-Gly-Gly-Gly-Lys(FITC)-Cys-NH₂ (wherePip=pipecolic acid) (SEQ ID NO:11); andGly-D-Phe-Pro-Arg-Ser-Gly-Gly-Gly-Gly-Lys(FITC)-Cys-NH₂ (SEQ ID NO: 12).

The new dyes of the invention can include one or more protective chainscovalently linked to the chromophore attachment moiety. Suitableprotective chains include polyhydroxyl compounds or other hydrophilicpolymers such as polyethylene glycol, methoxypolyethylene glycol,methoxypolypropylene glycol, copolymers of polyethylene glycol andmethoxypolypropylene glycol, polylactic-polyglycolic acid, poloxamer,polysorbate 20, dextran and its derivatives, starch and starchderivatives, and fatty acids and their derivatives. In certainembodiments of the invention, the chromophore attachment moiety ispolylysine and the protective chains are methoxypolyethylene glycols.

Synthesis of NIR Fluorescence (NIRF) Dyes

The synthetic pathways leading to eight new chromophores (referred to asNIR1, NIR2, NIR3, NIR4, NIR5, NIR6, NIR7, and NIR8) are illustrated inFIGS. 1, 2, and 3A.

The syntheses of NIR1 and NIR2 were carried out starting from1,1,2-trimethyl-benzindoleninium-1,3-disulfonate dipotassium salt, whichwas converted toN-ethyl-2,3,3-trimethyl-benzindoleninium-5,7-disulfonate 1. Reaction ofN-ethyl-2,3,3-trimethyl-benzindoleninium-5,7-disulfonate 1 withglutaconaldehydedianil hydrochloride and malonaldehyde dianilidehydrochloride, respectively, resulted in the intermediates 3 and 4.Intermediates 3 and 4 were stable at room temperature, even in aqueoussolution, and no significant decomposition was observed over two weeks.The asymmetrical fluorochrome dyes NIR1 and NIR2 were assembled byreacting intermediates 3 and 4, respectively, with5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin 2.

Similarly, the syntheses of NIR3 and NIR4 began with1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate 5, whichwas converted to intermediates 6 and 7 by the reaction withglutaconaldehydedianil hydrochloride and malonaldehyde dianilidehydrochloride, respectively. Like intermediates 3 and 4, intermediates 6and 7 were also stable at room temperature. Reaction of intermediates 6and 7 with 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin 2yielded the asymmetrical fluorochrome dyes NIR3 and NIR4, respectively.The final products were >98% pure as determined by HPLC.

Additionally, treatment of intermediates 3, 4, 8, and 9 with5-chloroacetamidomethyl-1,3,3-trimethyl-2methyleneindoline 10 affordedthe asymmetrical fluorochrome dyes NIR5, NIR6, NIR7, and NIR8respectively (see FIG. 3A). Intermediates 8 and 9 were prepared by thereaction between 1 11 and glutaconaldehydedianil hydrochloride andmalonaldehyde dianilide hydrochloride, respectively as shown in FIG. 3B.The chloroacetamino-containing cyanines NIR5-NIR8 were purified byreversed phase semi-preparative HPLC and were found to be approximately98% pure by reversed phase HPLC.

The synthesis of other NIRF dyes of the invention can be made reactingother indoleninium compounds with glutaconaldehydedianil hydrochloride,malonaldehyde dianilide hydrochloride, or other activated linker-formingcompounds. Other activated linker-forming compounds may include thosecompounds that form linkers containing one or more conjugated ringstructures, e.g., 12. The ring may contain e.g., four to eight membersand R¹⁵ can be hydrogen, substituted or unsubstituted alkyl, halogen, oran oxygen, nitrogen, sulfur, or phosphorus containing substituent. Fourand six-membered rings are preferred. For example, compound 13 can forma linker containing a six-membered ring.

The synthesis of various cyanine-type compounds is known in the art, asdescribed in Mishra et al., Chem. Rev., 100:1973-2011 (2000); Hamer, InThe Chemistry of Heterocyclic Compounds, Weissberger, Ed., Interscience:New York, 1964, Vol. 18; VankatRaman, The Chemistry of Synthetic Dyes,Academic Press: New York, 1952, Vol. II, p. 1143; Satapathy et al., J.Ind. Chem. Soc., 45:799 (1968); Mukherjee et al., J. Ind. Chem. Soc.,47:1121 (1970); Ficken, The Chemistry of Synthetic Dyes, Vankatraman,Ed., Academic Press: New York, 1971, Vol. IV, p. 211; Gamon et al.,Angew. Chem., 89:418 (1977); Dix et al., Angew. Chem., 90:8993 (1978);Mishra et al., J. Ind. Chem. Soc., 30A:886 (1991); Sahay et al., Ind. J.Chem. Soc., 27A:561 (1988); Mishra et al., Ind. J. Chem. Soc., 31B:118(1992); and Koraiem et al., Dyes Pigments, 15:89 (1991), which areincorporated herein by reference in their entireties. Given theinformation herein, it is within the ability of one of ordinary skill inthe art to synthesize the new chromophores without undueexperimentation.

Design of the New Cyanine Dyes Certain of the new cyanine dyes of theinvention bear two different heterocyclic ring systems, rendering themasymmetrical. Compounds NIR1 and NIR2 include both 3-ring and 2-ringheterocyclic systems. This design allows for fine-tuning of spectralproperties by changing the substitution group on the NIR fluorochromes.The asymmetrical design can also offer improvement in the typicallyserious self-aggregation of large planar dyes. The latter is ofparticular concern, since self-aggregating fluorochromes can be poorlysoluble, as is the case for indocyanine green (ICG). The new dyes'sconstant and large number of sulfonate groups further ensures andimproves their solubility.

Enzyme Activatable Imaging Probes

We previously developed a panel of biocompatible molecular probes forthe in vivo detection of specific protease activity, particularly forthose proteases that play key roles in different aspects of cancergrowth, metastases formation and angiogenesis (Tung et al., Canc. Res.,2000:4953-4958, 2000). We have now tested the new NIR dyes of theinvention as alternative reporters in this panel. The fluorochromes wereattached to a partially PEGylated graft copolymer (PGC) having apolylysine backbone as described in Bogdanov et al., Adv. Drug Deliv.Rev., 16:335-348 (1995). The probes were designed to have minimumfluorescence signal in their native states and to become highlyfluorescent after enzyme mediated release of fluorochromes, resulting insignal amplification. To reduce the initial fluorescence signal, a highlocal concentration of fluorochromes was desired to have significantself-quenching. Since the lysine residues on the PGC were only partiallyPEGylated, free amino groups on the unmodified lysine side chain couldbe used for fluorochrome attachment. Additional free lysine residueswere also needed for trypsin recognition. As a consequence, the numberof fluorochromes per polymer had to be optimized to maximize thefluorescence increase after enzymatic cleavage.

For this purpose, PGC probes were labeled with different numbers ofNIR2. Overall, seven conjugates were prepared, with an average of 0.2,0.8, 1.4, 2.4, 4.3, 5.7, and 7.0 NIR2 residues per PGC molecule,respectively. The white bars in FIG. 7 represent the fluorescent signalof the labeled polymers before trypsin treatment. An increase wasobserved in the signal from 0.2-0.8 dye molecules/polymer, while athigher dye/polymer ratio, considerable self-quenching was observed. Theblack bars in FIG. 7 correspond to the fluorescence signals obtainedafter 3 hours of tryptic cleavage. Maximum recovery was found for 4.3NIR2 per PGC. At this ratio, the fluorescence signal increased 5-fold in3 hours and 9-fold in 24 hours. Interestingly, recovery was lower whenmore NIR2 molecules were attached to the backbone. Without wishing to bebound by theory, the observed decrease may be due to there being fewerenzyme-accessible cleavage sites on the backbone when more dye moleculesare present.

Use of the New Cyanine Dyes for NIR Imaging

There are many biological processes that cannot be easily or directlymonitored with MRI, PET, or CT because key molecules in these processesare not distinguishable even in the presence of currently used contrastagents. NIP technology offers unique advantages for imaging ofpathology, because neither water nor many naturally occurringfluorochromes absorbs significantly in this region. Thus, NIR lightpenetrates tissues more efficiently than visible light or photons in theinfrared region. Exogenously added contrast agents can aid in thespecificity and sensitivity of disease detection. The new NIR contrastagents can be prepared in numerous forms, including as a free dye, analbumin-binding molecule, a targeting ligand, a quenched molecule, orother format.

Probe Design and Synthesis and Methods of Activation

Probe architecture, i.e., the particular arrangement of probecomponents, can vary, so long as the probe retains a chromophoreattachment moiety, and, optionally, spacers, and one or more (e.g., aplurality) of the new chromophores linked to the chromophore attachmentmoiety so that the optical properties of the chromophores are alteredupon activation of the imaging probe. For example, the activation sitescan be in the backbone itself or in side chains. Each chromophore can bein. a separate side chain, for example, or a pair of chromophores can bein a single side chain. In the latter case, an activation site can beplaced in the side chain between the pair of chromophores.

In some embodiments, the probe includes a polypeptide backbonecontaining only a small number of amino acids, e.g., 5 to 20 aminoacids, with chromophores attached to amino acids on opposite sides of aprotease cleavage (activation) site. Guidance concerning various probecomponents, including backbone, protective side chains, chromophores,chromophore attachment moieties, spacers, activation sites, andtargeting moieties is provided in the paragraphs below.

The chromophore attachment moiety design will depend on considerationssuch as biocompatibility (e.g., toxicity and immunogenicity), serumhalf-life, useful functional groups (for conjugating chromophores,spacers, and protective groups), and cost. Useful types of chromophoreattachment moieties, also referred to herein as “backbones,” includepolypeptides (polyamino acids), polyethyleneamines, polysaccharides,aminated polysaccharides, aminated oligosaccharides, polyamidoamines,polyacrylic acids, and polyalcohols. In some embodiments, the backboneconsists of a polypeptide formed from L-amino acids, D-amino acids, or acombination thereof. Such a polypeptide can be, e.g., a polypeptideidentical or similar to a naturally occurring protein such as albumin, ahomopolymer such as polylysine, or a copolymer such as a D-Tyr-D-Lyscopolymer. When lysine residues are present in the backbone, the ε-amino“groups” on the side chains of the lysine residues can serve asconvenient reactive groups for covalent linkage of chromophores andspacers. When the backbone is a polypeptide, the molecular weight of theprobe can be from 2 kD to 1000 kD, e.g., from 4 kD to 500 kD.

The chromophore attachment moieties can also be non-covalentlyassociated complexes, such as liposomes. Chromophores can be attached tolipids before or after liposome formation. When these complexes interactwith targets, the complexes can be activated, for example, withoutlimitation, by quenching, de-quenching, wavelength shift, fluorescenceenergy transfer, fluorescence lifetime change, and polarity change. Theprobes can be located entirely within such a liposome and releasedlocally with disruption of the liposome (such as with acoustic resonanceenergy imparted at ultrasound frequencies), or can be attached at thelipid surface.

A chromophore attachment moiety can be chosen or designed to have asuitably long in vivo persistence (half-life). Alternatively, a rapidlybiodegradable backbone such as polylysine can be used in combinationwith covalently linked protective chains. Examples of useful protectivechains include polyethylene glycol (PEG), methoxypolyethylene glycol(MPEG), methoxypolypropylene glycol, polyethylene glycol-diacid,polyethylene glycol monoamine, MPEG monoamine, MPEG hydrazide, and MPEGimidazole. The protective chains can also be block-copolymers of PEG anda different polymer such as a polypeptide, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Synthetic,biocompatible polymers are discussed generally in Holland et al.,Advances in Pharmaceutical Sciences, 6:101-164, 1992.

A useful backbone-protective chain combination ismethoxypoly(ethylene)glycol-succinyl-N-ε-poly-L-lysyine (PL-MPEG). Thesynthesis of this material, and other polylysine backbones withprotective chains, is described in Bogdanov et al., U.S. Pat. No.5,593,658 and Bogdanov et al., 1995, Advanced Drug Delivery Reviews,16:335-348.

Modifications to the chromophore attachment moiety can also be made toimprove delivery and activation. For example, graft copolymers can bemodified to improve the probes' biological properties and/or to improveactivation. For example, a 560 kD MFEG-PL graft copolymer randomlymodified with Cy5.5 to yield a cathepsin B-sensitive probe (as describedin the examples of U.S. Pat. No. 6,083,486) was further modified toyield a succinilated probe, i.e., the positive charges on the probe weremodified to neutral or negative charges by acetylation or succinilation,respectively, which demonstrated improved activation properties.

There are numerous other chemical modifications of polymers that can bemade, including changes in the charge of the polymer, changes in thepolymers' hydrophobic and hydrophilic properties, changes in the sizeand length of the polymer side chains, and addition of attractantsand/or binding moieties for enzymes. Examples of such modificationsinclude a large number of small molecules such as succinate, acetate,amino acids, phenyl, guanidinium, tetramethylguanidinium, methyl, ethyl,propyl, isopropyl, and benzyl.

Membrane translocation signals can also be added to the imaging probesto improve deliverability. Since many graft copolymers can enter variouscell types through fluid phase endocytosis, improvement of cellularuptake and assurance of cytoplasmic deposition of the imaging probe canbe achieved by attaching membrane translocation (or transmembrane)signal sequences. These signal sequences can be derived from a number ofsources including, without limitation, viruses and bacteria. Forexample, a Tat protein-derived peptide containing a caspase-3 sensitivecleavage site with thesequence—Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys(FITC)-Gly-Asp-Glu-Val-Asp-Gly-Cys(QSY7)-NH₂—(SEQ ID NO:3) has been shown to be efficiently internalized into cellsfor monitoring caspase-3 activity. The sequencesGly-Arg-Lys-Lys-Arg-Gln-Arg-Arg (SEQ ID NO:15) orGly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg (SEQ ID NO:16) can also be used.

Other targeting and delivery approaches can also be used such asfolate-mediated targeting (Leamon & Low, 2001, Drug Discovery Today,6:44-51), liposomes, transferrin, vitamins, carbohydrates and the use ofother ligands that target internalizing receptors, including, but notlimited to, somatostatin, nerve growth factor, oxytocin, bombesin,calcitonin, arginine vasopressin, angiotensin II, atrial nati-ureticpeptide, insulin, glucagons, prolactin, gonadotropin, and variousopioids. In addition, other ligands can be used that undergo anenzymatic conversion upon intracellular delivery that leaves theresulting conversion product trapped within the cell. Examples of suchligands include, for example, nitroheteroaromatic compounds that areirreversibly oxidized by hypoxic cells.

Intramolecular quenching by non-activated probes can occur by any ofvarious quenching mechanisms. Several mechanisms are known, includingresonance energy transfer between two chromophores. In this mechanism,the emission spectrum of a first chromophore should be very similar tothe excitation of a second chromophore, which is in close proximity tothe first chromophore. Efficiency of energy transfer is inverselyproportional to r⁶, where r is the distance between the quenchedchromophore and excited chromophore. Self-quenching can also result fromchromophore aggregation or excimer formation. This effect isconcentration-dependent. Quenching also can result from anonpolar-to-polar environmental change.

To achieve intramolecular quenching, several strategies can be applied.These include: (1) linking a second chromophore, as an energy acceptor,at a suitable distance from the first chromophore; (2) linkingchromophores to the backbone at high density, to induce self-quenching;and (3) linking polar chromophores in a vicinity of non-polar structuralelements of the backbone and/or protective chains. Partial or fullrecovery of the optical properties can be obtained upon cleavage of thechromophore from neighboring chromophores and/or from a particularregion, e.g., a non-polar region, of the probe.

The chromophore can be covalently linked to a chromophore attachmentmoiety or spacer using any suitable reactive group on the chromophoreand a compatible functional group on the chromophore attachment moietyor spacer. For example, a carboxyl group (or activated ester) on achromophore can be used to form an amide linkage with a primary aminesuch as the ε-amino group of the lysyl side-chain of polylysine.

In some embodiments of the invention, chromophores are linked to thechromophore attachment moiety through spacers containing activationsites. For example, oligopeptide spacers can be designed to containamino acid sequences recognized by specific proteases associated withtarget tissues. Some probes of this type accumulate in tumorinterstitium and inside tumor cells, e.g., by fluid phase endocytosis.By virtue of this accumulation, such probes can be used to image tumortissues, even if the enzyme(s) activating the probe are not tumorspecific.

In other embodiments of the invention, two paired chromophores inquenching positions are in a single polypeptide side chain containing anactivation site between the two chromophores. Such a side chain can besynthesized as an activatable module that can be used as a probe per se,or can be linked to a backbone or targeting moiety, e.g., an albumin,antibody, receptor binding molecule, synthetic polymer, orpolysaccharide. A useful conjugation strategy is to place a cysteineresidue at the N-terminus or C-terminus of the molecule, and then employSPDP for covalent linkage between the side chain of the terminalcysteine residue and a free amino group of the carrier or targetingmolecule.

In other embodiments, the probes are designed to be activated by variousenzymes, e.g., by cleavage. For example, prostate specific antigen(PSA), is a 33 kD chymotrypsin-like serine protease secreted exclusivelyby prostatic epithelial cells. Normally, this enzyme is primarilyinvolved in post-ejaculation degradation of the major human seminalprotein, and PSA concentrations are proportional to the volume ofprostatic epithelium. The release of PSA from prostate tumor cells,however, is about 30-fold higher than that from normal prostateepithelium cells. Damage to basal membrane and deranged tissuearchitecture allow PSA to be secreted directly into the extracellularspace and into the blood. Although high levels of PSA can be detected inserum, the serum PSA exists as a complex with al-antichymotrypsinprotein, and is proteolytically inactive. Free, uncomplexed, activatedPSA is present in the extracellular fluid from malignant prostatetissues, and PSA activity can be used as a marker for prostate tumortissue. Moreover, prostate tumor tissue is highly enriched in PSA;therefore, spacers containing the amino acid sequence recognized by PSAcan be used to produce an imaging probe that undergoes activationspecifically in prostate tumor tissue. An example of a PSA-sensitivespacer is His-Ser-Ser-Lys-Leu-Gln-Gly (SEQ ID NO:2). Other PSA-sensitivespacers can be designed using information known in the art regarding thesubstrate specificity of PSA. See, e.g., Denmeade et al., Cancer Res.57:49244930, 1997. These spacers can be included in the probe to makethem activatable by PSA.

Another example involves cathepsin D, an abundant lysosomal asparticprotease distributed in various mammalian tissues. In most breast cancertumors, cathepsin D is found at levels from 2-fold to 50-fold greaterthan levels found in fibroblasts or normal mammary gland cells. Thus,cathepsin D can be a useful marker for breast cancer. Spacers containingthe amino acid sequence recognized by cathepsin D can be used to producean imaging probe that undergoes activation specifically in breast cancertissue. An example of a cathepsin D-sensitive spacer is theoligopeptide: Gly-Pro-Ile-Cys-Phe-Phe-Arg-Leu-Gly (SEQ ID NO:1). Othercathepsin D-sensitive spacers can be designed using information known inthe art regarding the substrate specificity of cathepsin D. See, e.g.,Gulnik et al., FEBS Let., 413:379-384, 1997.

Another example involves matrix metalloproteinases (MMPs). Several MMPsare expressed in cancers at much higher levels than in normal tissue andthe extent of expression has been shown to be related to tumor stage,invasiveness, metastasis, and angiogenesis. MMP-2 (gelatinase) inparticular, has been identified as one of the key MMPs in theseprocesses, being capable of degrading type IV collagen, the majorcomponent of basement membranes. Based on these observations, severalcompanies have initiated the development of different M inhibitors totreat malignancies and other diseases involving pathologic angiogenesis.

The design of proteinase inhibitors has evolved over the last decade andnow largely relies on structure-based designs, the screening ofcombinatorial libraries, or employment of other combinatorial peptideapproaches. Through these efforts, a number of broad-spectrum and more“selective” MMP inhibitors have been described and are in clinicaltrials, while a number of agents are in preclinical development.Efficacy testing in animals has largely been measured as suppression oftumor growth based on tumor volume measurement following treatment andby assessment of histological and anti-angiogenic effects of MMPinhibitors in human tumor xenografts. However, differences in tumorgrowth usually do not reach statistical significance in murine modelsuntil 10-20 days after initiation of treatment. In a clinical setting,surrogate markers of treatment efficacy such as tumor regression, timeto recurrence or time to progression have been used because of the lackof more direct measures, although the limitations of such late endpointsare obvious.

MMP inhibitors can also be more effective when used in combination withchemotherapeutic agents. A specific molecular target-basedpharmacodynamic assessment of each therapeutic approach would thereforebe highly desirable (for estimating the relative contributions of eachagent and resulting synergies). For the reasons outlined above there isa need to directly detect and monitor proteinase activities in vivo inan intact tumor environment.

Spacers containing the amino acid sequence recognized by MMP-2 can beused to produce imaging probes that undergo activation specifically incancer tissue expressing MMP-2. An example of a MMP-2-sensitive spaceris the oligopeptide: GPLGVRGK(FITC)C—CH₂ (SEQ DNO:10). OtherMMP-2-sensitive spacers can be designed using information known in theart regarding the substrate specificity of MMP-2. In addition, other MMPprobes can be designed accordingly.

Various other enzymes can be exploited to provide probe activation(cleavage) in particular target tissues in particular diseases. Table 1provides information on several exemplary enzymes and associateddiseases (See Barrett et al., Handbook of Proteolytic Enzymes, AcademicPress, 1998).

Protease cleavage sites can be determined and designed using informationand techniques known in the art including using various compound andpeptide libraries and associated screening techniques (Turk et al.,Nature Biotech., 19:661-667, 2001).

In one embodiment of the present invention, when the chromophores arelinked directly to the backbone, probe activation can be achieved bycleavage of the backbone. High chromophore loading of the backbone caninterfere with backbone cleavage by activating enzymes such ascathepsins. Therefore, a balance between signal quenching andaccessibility of the backbone by probe-activating enzymes is important.For any given backbone-chromophore combination (when activation sitesare in the backbone), probes representing a range of chromophore loadingdensities can be produced and tested in vitro to determine the optimalchromophore loading percentage. TABLE 1 Enzyme-Disease AssociationsEnzyme Disease Reference Cathepsin B Cancer, Cardiovascular Weisslederet al., Nat. Disease, Arthritis, Biotech., 17: 375, 1999Neurodegenerative disease Cathepsin D Cancer Gulnik, FEBS Lett., 413:379, 1997 Cathepsin K Osteoporosis Atley et al., Bone, 26: Bone Cancer241-247, 2000 Cathepsin X Cancer Nägler et al., Biochemistry, 38:12648-12654, 1999 Cathepsin S Allergy, Asthma Riese et al., J. Clin.Invest., 101: 2351-2363, 1998 Caspases Apoptosis, Ischemia, Xiang etal., P.N.A.S., Arthritis, Neurodegenerative 93: 14559-14563, 1996disease, Cardiovascular Disease PSA Prostate Cancer Denmeade, CancerRes. 57: 4924, 1997 MMP's Cancer, Metastases, Verheijen, Biochem. J.Inflammation, Arthritis, 323: 603, 1997 Multiple Sclerosis, Maculardegeneration, Cardiovascular Disease CMV Viral Sardana, J. Biol. Chem.protease 269: 14337, 1994 Thrombin Blood clotting Rijkers, ThrombosisRes., 79: 491, 1995 Beta- Alzheimer Disease Berezovska et al., J. Biol.secretase Chem., 276: 30018-30023, (BACE) 2001 Urokinase CancerSchmalfeldt et al., Clin. plasminogen Cancer Res., 7: 2396, 2001activator

When the chromophores are linked to the backbone through activationsite-containing spacers, accessibility of the backbone byprobe-activating moieties is unnecessary. Therefore, high loading of thebackbone with spacers and chromophores does not significantly interferewith probe activation. For example, in such a system, every lysineresidue of polylysine can carry a spacer and chromophore, and everychromophore can be released by activating enzymes.

Accumulation of a probe in a target tissue can be achieved or enhancedby binding a tissue-specific targeting moiety to the probe. The bindingcan be covalent or non-covalent. Examples of targeting moieties includea monoclonal antibody (or antigen-binding antibody fragment) directedagainst a target-specific marker, a receptor-binding polypeptidedirected to a target-specific receptor, and a receptor-bindingpolysaccharide directed against a target-specific receptor.

Antibodies or antibody fragments can be produced and conjugated to theprobes described herein using conventional antibody technology (see,e.g., Folli et al., Cancer Res., 54:2643-2649, 1994; Neri et al., NatureBiotechnology, 15:1271-1275, 1997). Similarly, receptor-bindingpolypeptides, such as somatostatin peptide, and receptor-bindingpolysaccharides can be produced and conjugated to probes of thisinvention using known techniques. Other targeting and deliveryapproaches can also be used such as folate-mediated targeting approaches(Leamon et al., Drug Discovery Today, 6:44-51, 2001), and use ofliposomes, transferrin, vitamins, carbohydrates or other ligands thattarget internalizing receptors, including, but not limited to, nervegrowth factor, oxytocin, bombesin, calcitonin, arginine vasopressin,angiotensin II, atrial nati-uretic peptide, insulin, glucagons,prolactin, gonadotropin, and various opioids. In addition, other ligandscan be used that undergo an enzymatic conversion upon intracellulardelivery that leaves the resulting conversion product trapped in thecell. Examples of such ligands include nitroheteroaromatic compoundsthat are irreversibly oxidized by hypoxic cells.

In one embodiment, activation of the imaging probe can be achievedthrough phosphorylation or dephosphorylation of the probe.Phosphorylation is mediated through enzymes such as kinases, which areabundantly involved in signal transduction and function by catalyzingaddition of phosphate groups to serine, thronging, or tyrosine aminoacids. There are a number of different types of kinases including,without limitation, receptor tyrosine kinases, the Src family oftyrosine kinases, serine/thronging kinases, and the Mitogen-ActivatedProtein (MAP) kinases. In addition, many of these molecules areassociated with various disease states. Examples of kinases useful inthe present invention and their associated diseases are listed in Table2. TABLE 2 Kinase - Disease Associations Kinase Type Examples AssociatedDiseases Receptor Tyrosine Kinases 1. Epidermal Growth Factor 1. cancersof the digestive tract,    Receptor (EGFR)    breast and colorectalcancer 2. Her2/neu 2. breast cancer 3. Platelet-Derived Growth 3.fibroadenomas of the breast    Factor (PDGF) 4. Vascular Endothelial 4.angiogenesis    Growth Factor (VEGF) 5. Insulin receptor 5. diabetesmellitus Src family 1. Lyn 1. Wiskott-Aldrich syndrome 2. Fyn 2.Wiskott-Aldrich syndrome 3. Bruton's Tyrosine Kinase 3. X-Linkedammaglobulinemia    (BTK) Serine/Threonine 1. Protein Kinase C (PKC) 1.Diabetes-mellitus-related 2. cardiovascular 2. Alzheimer's syndrome   complications Mitogen-Activated Protein p38 Inflammation (MAP) kinases

Thus, in one embodiment of the present invention, phosphorylation isused to activate the probe. The phosphorylation of the serine,thronging, or tyrosine amino acids can cause attraction of thenegatively charged phosphate groups to the positively charged groups onthe opposite molecule, thus bringing the chromophores into aninteractive permissive position, causing changes in their opticalparameters, e.g., quenching, dequenching, wavelength shift, fluorescenceenergy transfer, fluorescence life time change, or polarity change. Themolecules can be fluorescence dyes, quenchers, and/or inducers (i.e.,compounds that cause fluorescence lifetime change or polarity change).Phosphorylation can also increase the local hydrophilicity, thusdecreasing the fluorescent resonance energy transfer betweenfluorochromes that is dependent upon local solvent concentration (e.g.,resulting in decreased quenching).

In other embodiments, the probes can be activated by utilizing an enzymethat removes or modifies a functional group (e.g., a phosphate group)located on the spacer of the probe. The probe is thus designed toincorporate a target sequence or chemical structure into a spacer thatis then modified or removed from the spacer to activate the probe. Inone example, a phosphate-ester metabolizing enzyme such as an alkalineor acid phosphatase is used. These enzymes hydrolyze phosphatemonoesters to an alcohol and an inorganic phosphate. Examples of enzymesuseful in the present invention include conjugates of calf intestinalalkaline phosphatase (CIP) and PTP1B and PTEN phosphatase inhibitors,the latter two of which have been developed for diabetes and gliomas,respectively.

Other forms of chemical modification such as methylation can also beutilized to activate the probes. Methylase enzymes covalently linkmethyl groups to adenine or cysteine nucleotides within restrictionenzyme target sequences, thus rendering them resistant to cleavage byrestriction enzymes. A methylation enzyme such as S-adenosylmethioninecan therefore be used to methylate a spacer of the imaging probe, thusrendering a quencher molecule resistant to restriction enzyme cleavage.Alternatively, a demethylase such as purified 5-MeC-DNA glycosylase canbe used to demethylate a spacer, thus allowing restriction enzymecleavage of a quenching molecule and the subsequent dequenching of thechromophore.

In other embodiments, probes containing mismatches or mutations in theirsequence are provided wherein the function of specific DNA repairenzymes is used to activate the probe. For example, a mismatch withinthe spacer of the imaging probe can result in the signal being quenched.Upon the correction of this mismatch by the appropriate DNA enzyme, aconformational change occurs, allowing the dequenching of the signal.There are several enzymes involved in DNA repair, including, withoutlimitation, poly ADP-ribose polymerase (PARP), DNA polymerases α, β, andΣ, and DNA ligase. Several human diseases result from deficiencies inDNA repair, including Ataxia-Telangiectasia, Xeroderma Pigmentosum,Cockayne Syndrome, and Santis-Caccione Syndrome. The loss of mismatchrepair enzyme function has also been associated with the earlydevelopment of many cancers.

Mutations can be inserted into the probe DNA in several different ways.For example, some methods of mutagenesis include: (1) use of degenerateoligonucleotides to create numerous mutations in a small DNA sequence;(2) spacer-scanning using nested deletions and complementary nucleotidesto insert point mutations throughout a sequence of interest; (3)spacer-scanning using oligonucleotide-directed mutagenesis; and (4) useof the polymerase chain reaction (PCR) to generate specific pointmutations.

Ubiquitin-specific target sequences can also be added to the probes,wherein the ubiquination of the target sequence allows for thechromophores to be brought into close proximity to permit energytransfer between the chromophores, thus activating the probe through anyof the mechanisms listed herein. Ubiquination is an important process inthe regulation of many biological processes, including angiogenesis andoxygen sensing. For example, the product of the von Hippel-Lindau (VHL)tumor suppressor gene (pVHL), whose loss of function contributes to VHLdisease and also contributes to 70% of renal cell carcinomas, has beenshown to directly promote degradation of Hypoxia-Indicuble-Factor (HIF)by ubiquination (Cockman et al., J., Biol. Chem., 275:25733-25741, 2000;Ohh et al., Nature Cell Biol., 2:423-427, 2000). Inhibitors of theubiquination pathway include Lactocystin and the Calpain I inhibitorLLnL (N-acetyl-Leu-Leu-Norleucinal) (Boriello et al., Oncogene,19(1):51-60, 2000).

In other embodiments, specific target binding sites are incorporatedinto the probes. These can include, without limitation, peptidesubstrates, enzyme binding sites, peptide sequences, sugars, RNA or DNAsequences, or other specific target binding sites or moieties. The probeis activated upon the binding of the target binding site, e.g., a changein the spectral properties of the chromophore occurs, for example, byadequate separation between the spacer and quencher. This is commonlyreferred to as a “molecular beacon.” Tyagi, Nature Biotech., 16:49,2000.

A number of specific peptide substrates including cathepsin B-specificpeptide substrates, MMP substrates, thrombin substrates and others areincluded in the probes of the present invention (see, e.g., Table 1).Examples of cathepsin B-specific substrates include RRK(FITC)C—CH₂ (SEQID NO:4), GRRK(FITC)C—CH₂ (SEQ ID NO:5), GRRRRK(FITC)C—CH₂ (SEQ IDNO:6), GRRGRRK(FITC)C—CH₂ (SEQ ID NO:7), GFGSVQ:FAGK(FITC)C—CH₂ (SEQ IDNO:8) (Peterson, Bioconjugate Chem., 10:553, 1999), andGFLGGK(FITC)C—CH₂ (SEQ ID NO:9), (Lu et al., Bioconjugate Chem.,12(1):129-133, 2001). An example of a MMP substrate isGly-Pro-Leu-Gly-Val-Arg-Gly-Lys(FITC)-Cys—CH₂ (SEQ ID NO:10). Examplesof thrombin-specific substrates (Rijkers D., Thrombosis Research 79:491,1995) include Gly-D-Phe-Pip-Arg-Ser-Gly-Gly-Gly-Gly-Lys(FITC)-Cys—CH₂(where Pip=pipecolic acid) (SEQ ID NO:11),Gly-D-Phe-Pro-Arg-Ser-Gly-Gly-Gly-Gly-Lys(FITC)-Cys—CH₂ (SEQ ID NO:12).

A monoclonal antibody (or antigen-binding antibody fragment) directedagainst a target-specific marker or a receptor-binding polypeptide orpolysaccharide directed against a target-specific receptor can also beused to activate the probe. Specific proteins include, but are notlimited to, G protein coupled receptors, nuclear hormone receptors suchas estrogen receptors, and receptor tyrosine kinases.

In other embodiments, enzymes that are capable of transferring thechromophore are used to activate the probe. Specific target sequencesthat are recognized by enzymes involved in recombination of DNA(recombinases) are incorporated into the probe. Upon recognition of thetarget site by the enzyme, the chromophore is transferred to anothermolecule (recombination) resulting in altered spectral properties of thechromophore or removal or alteration of the quencher from the spacer.Enzymes involved in recombination are well known in the art. Forexample, recombinases are involved in immunoglobulin (Ig) and T cellreceptor (TCR) gene rearrangements, a process involving therecombination of non-homologous gene segments, which occurs in immatureB and T cells. The genes that encode these recombinases have been clonedand identified as RAG-1 and RAG-2.

The probes can also be activated by incorporating into the probe targetsequences for enzymes involved in RNA splicing. This embodiment involvesincorporating an RNA splicing sequence (e.g., an intron segment) on thespacer portion of the probe, resulting in the alteration of the spacerlength. Activation is accomplished by changing the spectral propertiesof the chromophore, either by removing the quencher from the spacer ofthe probe, or by altering the quencher. Several methods of RNA splicingare known in the art. For example, splicing of introns from mRNA ismediated by a group of enzymes known as small nuclear RNAs (snRNAs),which complex together to form a splicosome. These enzymes splice RNA byprecisely breaking sugar-phosphate bonds at the boundaries of intronsand rejoining the free ends generated by intron removal into acontinuous mRNA molecule. There are also alternative splicing pathwaysthat allow for the formation of several different but related mRNAs thatin turn encode different but related proteins. For example, the thyroidhormone calcitonin and the calcitonin gene-related polypeptide found inhypothalamus cells are derived from the same pre-mRNA species, but dueto alternative splicing, result in two different, but related proteins.

The invention also features a fluorescent probes including afluorochrome attachment moiety and a plurality of fluorochromes whereinthe plurality of fluorochromes are chemically linked to the fluorochromeattachment moiety so that the spectral properties of the fluorochromesare altered upon “activation” of the fluorescent probe by an analyte.

An “analyte” can be a molecule or ion that binds to and activatesfluorescent probes. Such analytes include, but are not limited to, H⁺,Ca²⁺, Na⁺, Mg²⁺, Mn²⁺, Cl⁻, Zn²⁺, O₂, Fe²⁺, and K⁺ions, NO, and H₂O₂.

In one embodiment of the invention, analyte binding is used to activatethe probe. The binding of the analyte to the activation site causes ananalyte-induced conformational change, thus bringing the fluorochromesinto an interaction permissive position and causing changes in theiroptical parameters (e.g., quenching, dequenching, wavelength shift,fluorescence energy transfer, fluorescence life time change, or polaritychange). The molecules can be fluorescent dyes, quenchers, and/orinducers (i.e., a compound that causes a fluorescence lifetime change orpolarity change).

Peptides and polypeptides that selectively bind to analytes and undergoanalyte-induced conformational changes are known, including peptidesbased on zinc finger domains and calcium-binding EF-hand domains (See,e.g., Berg and Merckle, J. Am. Chem. Soc., 111:3759-3761, 1989; Krizel(et al., Inorg. Chen., 32:937-940, 1993; Krizek and Berg, Inorg. Chem.,31:2984-2986, 1992; Kim et al., J Biol. Inorg. Chem., 6:173-81, 2001;and U.S. Pat. No. 6,197,928). A single zinc finger domain is 25-30 aminoacids in length and has the consensus sequence(F/Y)-X-C-X₂₄—C-X₃-F-X₅-L-X₂-H—X₂₋₆(SEQ ID NO:13), where X is any aminoacid Berg, Acc. Chem. Res., 28:14-19, 1995).

A single EF-domain is a helix-loop-helix motif that usually has 12residues with the pattern, X-Z-X-Z-X-Z-X-Z-X-Z-Z-X (SEQ ID NO:14), whereX is an amino acid that participates in metal coordination, e.g.,histidine, glutamic acid, or aspartic acid, and Z represents theintervening amino acids, which can be any amino acid (bently et al.,Curr. Opin. Struct. Biol., 10:637-643, 2000).

Other peptide sequences and methods to design and screen for peptidesthat bind to specific analytes are also known (Bar-Or et al., Eur. J.Biochenm., 268:42-47, 2001; Enzelberger et al., J. Chromatogr. A.,10:83-94, 2000; Fattorusso et al., Biopolymers, 37:401-410, 1995; Bonomoet al., Chemistry, 6:4195-4202, 2000; Ashraf et al., Bioorg. Med. Chem.,10: 1617-1620, 2000; Zoroddu et al., J. Inorg. Biochem., 84:47-54, 2001;Mukhejee et al., Indian Chem. Soc., 68:639-642, 1991; Hulsbergen et al.,Recl Trav. Chim. Pays-Bas, 112:278-286, 1993; Ama et al., Bull Chem.Soc. Japan, 62:3464-3468, 1989; U.S. Pat. No. 6,083,758 and U.S. Pat.No. 5,928,955).

In another embodiment, probes can be activated by changes in H+ ionconcentration or pH changes. Probes can be designed to contain spacersthat are cleaved when physiological pH values are lowered. Examples ofsuch spacers include alkylhydrazones, acylhydrazones, arylhydrazones,sulfonylhydrazones, imines, oximes, acetals, ketals, and orthoesters.

The methods of analyte activation described herein can be used to detectand/or evaluate many diseases or disease-associated conditions. Theredistribution of analytes such as potassium, sodium, and calcium isoften indicative of certain physiological processes and diseasesincluding hypoxia and ischemia (e.g., cerebro-vascular ischemia due tostroke, embolism or thrombosis; ischemia of the colon; vascular ischemiadue to coronary artery disease of heart disease; ischemia due tophysical trauma or poisons; ischemia associated with encephalopathy; andrenal ischemia). In addition, tumors are characterized by low pH valuesin comparison with normal tissue, as well as inflammation, particularlyinflammation caused by foreign pathogens.

In another embodiment, a quencher molecule is used to quench the initialsignal. Prior to activation, the quencher molecule is situated such thatit quenches the optical properties of the reporter molecule (i.e.,chromophore). Upon activation, the reporter molecule is de-quenched. Byadopting these activated and unactivated states in a living animal orhuman, the reporter molecule and quencher molecule located on the probewill exhibit different signal intensities, depending on whether theprobe is active or inactive. It is therefore possible to determinewhether the probe is active or inactive in a living organism byidentifying a change in the signal intensity of the reporter molecule,the quencher molecule, or a combination thereof. In addition, becausethe probe can be designed such that the quencher molecule quenches thereporter molecule when the probe is not activated, the probe can bedesigned such that the reporter molecule exhibits limited signal untilthe probe is either hybridized or digested.

There are a number of quenchers available and known to those skilled inthe. art including, but not limited to, DABCYL, QSY-7 (Molecular Probes,Inc., OR), QSY-33 (Molecular Probes, Inc., OR), and fluorescence dyessuch as Cy5 and Cy5.5 pare (Schobel, Bioconjugate 10:1107, 1999).

An additional method of detection includes two distinct fluorochromes(termed “fluorochrome1” and “fluorochrome2”) that are spatially near oneanother such that fluorescent resonance energy transfer (FRET) takesplace. Thus, initially, excitation at fluorochromel's excitationwavelength results in emission at fluorochrome2's emission wavelengthsecondary to FRET. Activation of the probe can be determined in thisembodiment as loss of signal at fluorochrome2's emission wavelength withexcitation at fluorochrome1's excitation wavelength. Signal increase atfluorochromel's emission wavelength after excitation at fluorochrome1'sexcitation wavelength can aid the determination of activation in thiscase. Emission at fluorochrome2's emission wavelength after excitationat the fluorochrome2's excitation wavelength can also be used todetermine local probe concentration.

Alternatively, the FRET method can be used to determine activation ofprobes when two components are brought into proximity after enzymaticactivity (e.g., ubiquination), such that fluorochromel andfluorochrome2, which are initially spatially separated, are subsequentlyspatially near enough to each other for FRET to take place. Thus,activation is detected by exciting at fluorochrome1's excitationwavelength and recording at fluorochrome2's emission wavelength.

In Vitro Probe Testing

After an imaging probe is designed and synthesized, it can be tested invitro to verify a requisite level of signal before activation.Preferably, this can be done by obtaining signal values for parameterssuch as quenching, de-quenching, wavelength shift, fluorescence energytransfer, fluorescence lifetime change, and polarity change of thefluorochrome-containing probe, in a dilute, physiological buffer. Thesevalues are then compared with the corresponding signal values obtainedfrom an equimolar concentration of free chromophore in the same buffer,under the same chromophore-measuring conditions. Preferably, thiscomparison is done using a series of dilutions, to verify that themeasurements are taking place on a linear section of the signal valuevs. chromophore concentration curve.

The molar amount of a chromophore on a probe can be determined by one ofordinary skill in the art using any suitable technique. For example, themolar amount can be determined readily by near infrared absorptionmeasurements. Alternatively, the molar amount can be determined readilyby measuring the loss of reactive linking groups on the backbone orspacer, e.g., decrease in ninhydrin reactivity due to loss of aminogroups.

In another procedure, the chromophore signal emittance is measuredbefore and after treatment with an activating agent, e.g., an enzyme. Ifthe probe has activation sites in the backbone (as opposed to inspacers), de-quenching should preferably be tested at various levels ofchromophore loading. “Loading” in this context refers to the percentageof possible chromophore linkage sites on the backbone actually occupiedby chromophores.

In addition, cells grown in culture can routinely be used to test theimaging probes of the present invention. Free probe molecules in cellculture medium should be non-detectable by fluorescence microscopy,while cellular uptake should result in probe activation and afluorescence signal from probe-containing cells. Microscopy of culturedcells can thus be used to verify that activation takes place when cellstake up a probe being tested. Microscopy of cells in culture is also aconvenient means for determining whether activation occurs in one ormore subcellular compartments.

The compositions and methods of the present invention can be used incombination with other imaging compositions and methods. For example,the methods of the present invention can be used in combination withtraditional imaging modalities such as CT and MRI.

The imaging methods of the present invention can also be combined withtherapeutic methods. For example, an immediate anti-tumor therapy can beemployed if the probes of the present invention detect a tumor.

In Vivo Near Infrared Imaging

Although the invention involves novel imaging probes, general principlesof fluorescence, optical image acquisition, and image processing can beapplied in the practice of the invention. For a review of opticalimaging techniques, see, e.g., Alfano et al., Ann. NY Acad. Sci.,820:248-270, 1997.

An imaging system useful in the practice of this invention typicallyincludes three basic components: (1) a near infrared light source, (2)apparatus for separating or distinguishing emissions from light used forchromophore excitation, and (3) a detection system.

The light source provides monochromatic (or substantially monochromatic)near infrared light. The light source can be a suitably filtered whitelight, e.g., bandpass light from a broadband source. For example, lightfrom a 150-watt halogen lamp can be passed through a suitable bandpassfilter commercially available from Omega Optical (Brattleboro, Vt.). Insome embodiments, the light source is a laser. See, e.g., Boas et al.,Proc. Natl. Acad. Sci. USA 91:4887-4891, 1994; Ntziachristos et al.,Proc. Natl. Acad. Sci. USA 97:2767-2772, 2000; Alexander, J. Clin. LaserMed. Surg. 9:416-418, 1991. Information on near infrared lasers forimaging can also be found on the Internet (e.g., at http://www.imds.com)and various other well-known sources.

A high pass or bandpass filter (700 nm) can be used to separate opticalemissions from excitation light. A suitable high pass or bandpass filteris commercially available from Omega Optical. In the case of quantumdots, a single excitation wavelength can be used to excite multipledifferent fluorochromes on a single probe or multiple probes (withdifferent activation sites), and spectral separation with a series ofbandpass filters, diffraction grating, or other means can be used toindependently read the different activations.

In general, the light detection system can includelight-gathering/image-forming and light-detection/image-recordingcomponents. Although the light-detection system can be a singleintegrated device that incorporates both components, thelight-gathering/image-forming and light-detection/image-recordingcomponents will be discussed separately. However, a recording device maysimply record a single (time varying) scalar intensity instead of animage. For example, a catheter-based recording device can recordinformation from multiple sites simultaneously (i.e., an image), or canreport a scalar signal intensity that is correlated with location byother means (such as a radio-opaque marker at the catheter tip, viewedby fluoroscopy).

A particularly useful light-gathering/image-forming component is anendoscope. Endoscopic devices and techniques that have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. Photochem. Photobiol. B 52:131-135, 1999),ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132, 1997), colon(Mycek et al., Gastrointest. Endosc. 48:390-394, 1998; Stepp et al.,Endoscopy 30:379-386, 1998) bile ducts (Izuishi et al.,

Hepatogastroenterology 46:804-807, 1999), stomach (Abe et al., Endoscopy32:281-286, 2000), bladder (Kriemair et al., Urol. Int. 63:27-31, 1999;Riedl et al., J., Endourol. 13:755-759, 1999), and brain (Ward, J. LaserAppl. 10:224-228, 1998) can be employed in the practice of the presentinvention.

Other types of light gathering components useful in the invention arecatheter-based devices, including fiber optic devices. Such devices areparticularly suitable for intravascular imaging. See, e.g., Tearney etal., Science 276:2037-2039, 1997; Boppart et al., Proc. Natl. Acad. Sci.USA 94:4256-4261, 1997.

Still other imaging technologies, including phased array technology(Boas et al., Proc. Natl. Acad. Sci. USA 91:48874891, 1994; Chance, Ann.NY Acad. Sci. 838:29-45, 1998), diffuse optical tomography (Cheng etal., Optics Express 3:118-123, 1998; Siegel et al., Optics Express4:287-298, 1999), intravital microscopy (Dellian et al., Br. J. Cancer82:1513-1518,2000; Monsky et al, Cancer Res. 59:4129-4135, 1999;Fukumura et al., Cell 94:715-725, 1998), and confocal imaging (Korlachet al., Proc. Natl. Acad. Sci. USA 96:8461-8466, 1999; Rajadhyaksha etal., J. Invest. Dermatol. 104:946-952, 1995; Gonzalez et al., J. Med.30:337-356, 1999) can be employed in the practice of the presentinvention.

Any suitable light-detection/image-recording component, e.g.,charge-coupled device (CCD) systems or photographic film, can be used inthe invention. The choice of light-detection/image-recording componentwill depend on factors including type of light gathering/image formingcomponent being used. Selecting suitable components, assembling theminto a near infrared imaging system, and operating the system is withinthe ability of a person of ordinary skill in the art.

In some embodiments of the invention, two (or more) probes containing:

-   (1) chromophores that emit optical signals at different near    infrared wavelengths, and-   (2) activation sites recognized by different enzymes, e.g.,    cathepsin D and MMP2, are used simultaneously. This allows    simultaneous evaluation of two (or more) biological phenomena.

In some embodiments of the invention, an additional chromophore thatemits light at a different near infrared wavelength is attached to theprobe that is not in an optical-quenching interaction-permissiveposition. Alternatively, two chemically similar probes, one activatableand one non-activatable, each labeled with a different chromophore, canbe used. By using the ratio of activatable to non-activatable probefluorescence, the activity of enzymes can be determined in a manner thatis corrected for the ability of tissues to accumulate variable amountsof these probes. Both of these approaches can be used to monitordelivery of the probe, to track the probe, to calculate doses, and toserve as an internal standard for calibration purposes.

Pharmaceutically acceptable carriers, adjuvants, and vehicles can beused with the compounds of this invention. Useful carriers, adjuvants,and vehicles include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins such as albumin, buffersubstances such as phosphate, glycine, sorbic acid, potassium sorbate,tris(hydroxymethyl)amino methane (“TRIS”), partial glyceride mixtures offatty acids, water, salts or electrolytes, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polypropylene block co-polymers,sugars such as glucose, and suitable cryoprotectants.

The probes of the invention can be administered in the form of a sterileinjectable preparation. This preparation can be prepared by thoseskilled in the art of such preparations according to techniques known inthe art. The possible vehicles or solvents that can be used to makeinjectable preparations include water, Ringer's solution, and isotonicsodium chloride solution, and 5% D-glucose solution (D5W). In addition,oils such as mono- or di-glycerides and fatty acids such as oleic acidand its derivatives can be used.

The probes of the present invention can be administered orally,parenterally, by inhalation, topically, rectally, nasally, buccally,vaginally, or via an implanted reservoir. The term “parenteraladministration” includes intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intraperitoneal,intracisternal, intrahepatic, intralesional, and intracranial injectionor infusion techniques. The probes can also be administered viacatheters or through a needle to any tissue.

For ophthalmic use, the probes of the invention can be formulated asmicronized suspensions in isotonic, pH-adjusted, sterile saline.Alternatively, the compositions can be formulated in ointments such aspetrolatum.

For topical application, the probes can be formulated in a suitableointment, such as petrolatum. Transdermal patches can also be used.Topical application for the lower intestinal tract or vagina can beachieved by a suppository formulation or enema formulation.

The formulation of the probe can also include an antioxidant or someother chemical compound that prevents or reduces the degradation of thebaseline fluorescence, or preserves the fluorescence properties,including, but not limited to, quantum yield, fluorescence lifetime, andexcitation and emission wavelengths. These antioxidants or otherchemical compounds can include, but are not limited to, melatonin,dithiothreitol (dTT), defroxamine (DFX), methionine, and N-acetylcysteine.

Dosing of the new chromophores and probes will depend on a number offactors including the instruments' sensitivity, as well as a number ofsubject-related variables, including animal species, age, body weight,mode of administration, sex, diet, time of administration, and rate ofexcretion.

Prior to use of the invention or any pharmaceutical composition of theinvention, the subject can be treated with an agent or regimen toenhance the imaging process. For example, a subject can be put on aspecial diet prior to imaging to reduce any auto-fluorescence orinterference from ingested food, such as a low pheophorbide diet toreduce interference from fluorescent pheophorbides that are derived fromsome foods, such as green vegetables. Alternatively, a cleansing regimencan be used prior to imaging, such as those cleansing regimens that areused prior to colonoscopies and include use of agents such as Visiciol.

The subject (patient or animal) can also be treated with pharmacologicalmodifiers to improve image quality. For example, using low doseenzymatic inhibitors to decrease background signal relative to targetsignal (secondary to proportionally lowering enzymatic activity ofalready low-enzymatic activity normal tissues to a greater extent thanenzymatically-active pathological tissues) can improve thetarget-to-background ratio during disease screening. As anothernon-limiting example, pretreatment with methotrexate to relativelyincrease uptake in abnormal tissue (i.e., metabolically active cancers)in conjunction with folate-based targeted delivery can be employed.

The invention is further described in the following examples, which arenot intended to limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Synthesis and Characterization of New ChromophoresNIR1-4

Fluorochrome dyes NIR1, NIR2, NIR3, and NIR4 were synthesized accordingto the following procedure.

Starting Materials: 1,1,2-Trimethylbenzindoleninium 1 ,3-disulfonatedipotassium salt,5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin2,1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate, and5-chloroacetamido-1,3,3-trimethyl-2-methyleneindoli 10 were synthesizedaccording to literature methods (Mujumdar et al., Bioconjug. Chem.,7:356-362, (1996); Terpetschnig et al., Anal. Biochem., 217:197-204(1994); Mujemdar et al., Bioconug. Chem., 4:105-111 (1993), and Gale, D.J.; Wilshire, J. F. K. J. Soc. Dyers Colour. 1974, 90, 97-100,respectively). All compounds were used in crude form.

N-ethyl-2,3,3-trimethyl-benzindoleninium-5,7-disulfonate 1: 4.7 g of1,1,2-trimethylbenzindoleninium 1,3-disulfonate dipotassium salt 8 ml ofethyl iodide (Aldrich Chemical Co., Milwaukee, Wis.), and 50 ml of1,2-dichlorobenzene (Aldrich) were added to a round bottom flask. Themixture was heated under an argon atmosphere at 90° C. for 12 hours andthen at 125° C. for another 10 hours. After cooling the mixture to roomtemperature, the solvent was decanted and the solid residue was washedthree times with an acetone/ether mixture. The solid was recovered byfiltration and dried under vacuum to result in 4.1 g of crudeN-ethyl-2,3,3-trimethyl-benzindoleninium-5,7-disulfonate 1.

Intermediate 3: 1.92 g ofN-ethyl-2,3,3-trimethylbenzindoleninium-5,7-disulfonate 1 1.12 g ofglutaconaldehydedianil hydrochloride (TCI America, Portland, Oreg.), 20ml of acetic anhydride (Aldrich), and 5 ml of glacial acetic acid(Aldrich) were added to a 50 ml round bottom flask, and the resultingmixture was heated at 120° C. for 3 hours. After cooling the mixture, itwas added to ethyl acetate (Aldrich), causing a solid to precipitate.The solid was recovered by filtration, and then washed twice with ethylacetate and dried under vacuum to yield 2.2 g crude intermediate 3.

NIR1: A mixture of 0.60 g of intermediate 3, 0.33 g of5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin 2, 0.49 g ofpotassium acetate (Aldrich), 12 ml of acetic anhydride, and 5 ml ofglacial acetic acid was stirred and heated at 120° C. under an argonatmosphere for 30 minutes. After cooling to room temperature, themixture was poured into 200 ml of ethyl acetate, causing a solid toprecipitate. The precipitated solid was collected by centrifugation andthen dried to result in 0.86 g of crude NIR1. The crude product wasfurther purified by reverse phase HPLC to give 14% (based on the crudeproduct) of pure NIR1. ¹H NMR (D₂O) (400 MHz FT-NMR spectrometer): δ1.24 (6H, s), 1.28 (3H, t), 1.54 (6H, s), 1.80 (4H, broad m), 2.90 (2H,broad t), 3.86 (2H, broad), 4.12 (2H, broad q), 5.76 (1H, broad), 6.03(2H, broad), 7.04 (2H, broad), 7.21 (1H, d), 7.33 (1H, broad), 7.53 (1H,broad), 7.58 (1H, s), 7.65 (1H, d), 7.73 (1H, d), 8.19 (1H, s), 8.52(1H, s), 8.71 (1H, d).

(NIR5): A mixture of 170 mg of intermediate 3, 36 mg of5-chloroacetamido-1,3,3-trimethyl-2-methyleneindoline 10, 5 mL of aceticanhydride, 2.5 mL of glacial acid, and 140 mg of potassium acetate werestirred and heated at 115° C. under argon atmosphere for 18 minutes.After cooling to room temperature the mixture was poured into 80 mL ofethyl acetate. The precipitate was collected by centrifugation and driedto result in 120 mg of crude NIR5. The crude product was furtherpurified by reverse phase HPLC to give 7% (based on the crude product)of pure product. ¹H NMR (D₂O/CD₃CN, 1:1): δ 1.31 (3H, t), 1.59 (6H, 6),1.88 (6H, s), 3.47 (3H, s), 4.11 (2H, broad q), 4.16 (2H, s), 6.13-6.19(2H, m), 6.43-6.49 (2H, broad), 7.17 (1H, d), 7.47 (2H, d), 7.65 (2H,d), 7.77-7.90 (2H, m), 8.2 (1H, s), 8.60 (1H, s), 8.82 (1H,d).

Intermediate 4: 1.70 g ofN-ethyl-2,3,3-trimethylbenzindoleninium-5,7-disulfonate 1 0.93 g ofmalonaldehyde dianilide hydrochloride (TCI America), 20 ml of aceticanhydride, and 5 ml of glacial acetic acid were added to a 50 ml roundbottom flask. The resulting mixture was then heated at 120° C. for 3hours. Upon cooling, the mixture was poured into ethyl acetate, causinga solid to precipitate. After recovery by filtration, the solidprecipitate was washed twice with ethyl acetate and dried under vacuumto yield 1.5 g crude intermediate 4.

NIR2: A mixture of 0.58 g of intermediate 4, 0.35 g of5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin 2, 0.49 g ofpotassium acetate, 12 ml of acetic anhydride, and 5 ml of glacial aceticacid was stirred and heated at 120° C. under argon atmosphere for 30minutes. After cooling to room temperature, the mixture was poured into200 ml of ethyl acetate, causing a solid to precipitate. The solid wascollected by centrifugation and dried to yield 0.9 g of crude NIR2. Thecrude product was then purified by reversed phase HPLC to give 21%(based on the crude product) of pure NIR2. ¹ NMR(D₂O): δ 1.19 (3H, t),1.24 (6H, s), 1.52 (6H, s), 1.80 (4H, broad m), 2.90 (2H, broad t), 3.90(2H, broad m), 4.02 (2H, broad q), 5.82 (1H, broad d), 5.85 (1H, broadd), 6.14 (1H, t), 7.08 (1H, d), 7.56 (1H, s), 7.61 761-7.77 (4H, m) 8.19(1H, s), 8.51 (1H, s), 8.70 (1H, d).

NIR6: A mixture of 121 mg of intermediate 4, 49 mg of 10, 5 mL of aceticanhydride, 2 mL of glacial acid, and 110 mg of potassium acetate werestirred and heated at 120° C. under argon atmosphere for 20 minutes.After cooling to room temperature the mixture was poured into 80 mL ofethyl acetate. The precipitate was collected by centrifugation and driedto result in 100 mg of crude NIR5. The crude product was then purifiedby reversed phase HPLC with a yield of 14% of pure product (based on thecrude product). ¹H NMR (D2O/CD₃CN, 2:1): 67 1.24 (3H, s), 1.43 (6H, s),1.71 (6H, s), 3.45 (3H, broad s), 3.91 (2H, s), 4.03 (2H, broad q),5.94-6.03 (2H, broad m), 6.31 (1H, broad m), 7.11 (1H, d), 7.44 (1H,dd), 7.49 (1H, s), 7.75-7.95 (2H, m), 8.21 (1H, s), 8.55 (1H, s), 8.79(1H, d).

Intermediate 6: In a 50 ml round flask were placed 0.80 g of1-(4-sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate 5, 0.50 gof glutaconaldehydedianil hydrochloride, 10 ml of acetic anhydride, and5 ml of glacial acetic acid. The resulting mixture was then heated at120° C. for 3 hours. Upon cooling, the mixture was poured into ethylacetate, causing a solid to precipitate. After filtration, the solidprecipitate was washed twice with ethyl acetate and dried under vacuumto yield 0.88 g crude intermediate 6.

NIR3: A mixture of 0.88 g of intermediate 6, 0.55 g of5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin 2, 0.85 g ofpotassium acetate, 12 ml of acetic anhydride, and 5 ml of glacial acidwere stirred and heated at 120° C. under argon atmosphere for 30minutes. After cooling to room temperature, the mixture was poured into200 ml of ethyl acetate, causing a solid to precipitate. The precipitatewas collected by centrifugation and dried to yield 0.9 g of crude NIR3.The crude product was then purified by reverse phase HPLC to give 6.7%(based on the crude product) of pure NIR3. ¹H NMR (D₂O): δ 1.36 (6H, s),1.42 (6H, s), 1.56-1.81 (6H, broad m), 1.81-1.89 (2H, broad m),2.84-2.90 (4H, m), 3.92 (2H, broad t), 4.06 (2H, broad t), 5.85 (1H,broad d), 6.10 (2H, broad), 6.28 (1H, broad t), 7.17 (2H, broad m), 7.26(1H, d), 7.45 (1H, broad), 7.53 (1H, broad), 7.67-7.77 (4H, m).

Intermediate 7: 0.80 g of1-(4sulfonatobutyl)-2,3,3-trimethylindoleninium-5-sulfonate 5, 0.45 g ofmalonaldehyde dianilide hydrochloride, 10 ml of acetic anhydride, and 5ml of glacial acetic acid were added to a 50 ml round bottom flask. Theresulting mixture was then heated at 120° C. for 3 hours. Upon cooling,the mixture was poured into ethyl acetate, causing a solid toprecipitate. After filtration, the solid was washed twice with ethylacetate and dried under vacuum to yield 0.74 g crude intermediate 7.

NIR4: A mixture of 0.74 g of intermediate 7, 0.45 g of5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenin 2, 0.70 g ofpotassium acetate, 12 ml of acetic anhydride, and 5 ml of glacial aceticacid were stirred and heated at 120° C. under argon atmosphere for 30minutes. After cooling to room temperature, the mixture was poured into200 ml of ethyl acetate, causing a solid to precipitate. The solid wascollected by centrifugation and dried to yield 0.9 g of crude NIR4. Thecrude product was then purified by reverse phase HPLC to give 8.1%(based on the crude product) of pure NIR4. ¹H NMR (D²O): 1.29 (6H, s),1.39 (6H, s), 1.71-1.78 (6H, broad m), 1.84-1.88 (2H, broad m),2.79-2.87 (4H, m), 3.92 (2H, broad t), 4.04 (2H, broad t). 5.82 (1H, d),6.06 (1H, d), 6.23 (1H, t), 7.20 (1H, d), 7.25 (1 H,d) 7.60-7.75 (6H,m).

Intermediate 11: Into a 250 mL round-bottomed flask were placed 2.2 g of1,1,2-trimethylbenzindolenium 1,3-disulfonate dipotassium salt, 1.4 mLof 1,4-butane sultone, and 20 mL of 1,2-dichlobenzene. The reactionmixture was heated under argon atmosphere at 125° C. for 24 hours. Afterbeing cooled to room temperature, the solvent was decanted and the solidwas washed three times with acetone. The solid was filtered off anddried under vacuum to yield 1.82 g of crude product 11 (FIG. 3B).

Intermediate 8: In a 200 mL round-bottomed flask were placed 0.98 g of11 (FIG. 3B), 0.56 g of glutaconaldehyde dianil hydrochloride (TCI), 10mL of acetic anhydride, and 4 mL of acetic acid. The mixture was heatedat 125° C. for 3 hours. The mixture was then precipitated from ethylacetate upon cooling. After filtration, the solid was washed twice withethyl acetate and dried under vacuum to yield 1.12 g of crudeintermediate 8 (FIG. 3B). UV (H₂ O): 495 nm.

NIR7: A mixture of 160 mg of intermediate 8 (FIG. 3B), 34 mg ofintermediate 10, 5 mL of acetic anhydride, 2.5 mL of glacial acid and108 mg of potassium acetate were stirred and heated at 125° C. underargon atmosphere for 25 minutes. After cooling to room temperature, themixture was poured into 80 mL of ethyl acetate. The precipitate wascollected by centrifugation and dried to result in 110 mg of crude NIR7(FIG. 3A). The crude product was then purified by reversed phase HPLC toyield 6% of pure product (based on the crude product). ¹H NMR(D)₂O/CD₃CN, 2:1): δ 1.80 (6H, s), 2.04 (6H, s), 2.09 (4H, broad m),3.10 (3H, broad t), 3.78 (2H, broad s), 4.22 (2H, broad) 4.27 (2H, s),6.40-6.55 (1H, broad d), 6.55-6.75 (2H,broad), 746 (1H, broad), 767 (1H,dd), 7.75-784 (1H, broad), 785, (1H, s), 7.90 (1H, d), 7.92-8.10 (2H,broad m), 8.44 (1H, s), 8.83 (1H, s), 9.02 (1H, d)

Intermediate 9: In a 200 mL round-bottomed flask were placed 0.90 g of11 (FIG. 3B), 0.49 g of malonaldehyde dianil hydrochloride (TCI America,Portland, Oreg), 10 mL of acetic anhydride, and 4 mL of acetic acid. Themixture was heated at 120° C. for 3 hours, then precipitated from ethylacetate upon cooling. After filtration, the solid was washed twice withethyl acetate and dried under vacuum to yield 1.08 g of crudeintermediate 9 (FIG. 3B). UV (H₂ O): 485 nm.

NIR8. A mixture of 150 mg of intermediate 9, 30 mg of intermediate 10, 5mL of acetic anhydride, 2 mL of glacial acid, and 100 mg of potassiumacetate were stirred and heated at 125° C. under argon atmosphere for 25minutes. After cooling to room temperature the mixture was poured into80 mL of ethyl acetate. The precipitate was collected by centrifugationand dried to result in 120 mg of crude NIR8 (FIG. 3A). The crude productwas then purified by reversed phase HPLC to yield 9% of pure product(based on the crude product). ¹H NMR (D₂O/CD₃CN, 2:1): δ 1.84 (6H, s),2.08 (6H, s), 2.10 (4H, broad m), 3.11 (3H, broad t), 3.78 (2H, broads), 4.27 (2H, s), 4.32 (2H, broad), 6.36- (1H, broad d), 6.45 (1H, broadd), 6.64-6.71 (1H, broad m), 7.46 (1H, d), 7.68 (1H, dd), 7.84 (1H, s),7.96 (1H, d), 8.19-8.35 (2H, broad m). 8.47 (1H, s), 8.86 (1H, s), 9.05(1H, d)

Example 2 Determination of Extinction Coefficients of Fluorochrome Dyes

All of the new NIR fluorochrome dyes were purified twice by preparativeHPLC, using a preparative HPLC instrument (Rainin, Woburn. Mass.) with aC18-RP preparative column (Vydec, Hesperia, Calif.) (flow rate=6 ml/min;eluant A, water with 0.1% TFA; eluant B, 90% of acetonitrile and 10% ofeluant A; starting at 90% A for 5 min and then a linear gradient over 40min to 50% A). The instrument's dual HPLC detector was set at 240 and360 nm. The dyes were collected, and solvent was removed using aspeed-vac concentrator (Savant, Holbrook, N.Y.).

The K+ions of the potassium salts were replaced with H+ to generate thecorresponding free acids by ion-exchange chromatography (cation-resin,Dowex-50, 8% cross-link, 100-200 mesh).

About 20 mg of each fluorochrome dye was dissolved in 100 ml ofdeionized water. The absorbance was measured individually in threedilutions of the stock solution in deionized water or in 95% ethanol,using a Hitachi U-3000 spectrophotometer to determine the extinctioncoefficient. The fluorescence emission maxima and intensities of thedyes were obtained using a Hitachi F-4500 fluorophotometer, using dilutesolutions in water and exciting at both the main absorption peak as wellthe short-wavelength shoulder of the main absorption peak. In the casesof NIR2, NIR4, NIR6, and NIR8, the quantum yields were calculatedrelative to a standard solution of the commercially availablefluorochrome Cy5.5 (Amersham-Pharmacia, Piscataway, N.J.) with quantumyield of 0.29. The calculations for NIR1, NIR3, NIR5, and NIR7 wereperformed relative to a standard solution of another commerciallyavailable fluorochrome, Cy7 (Amersham-Pharmacia), with a quantum yieldof 0.28.

Example 3 Activation of Fluorochrome Dyes

The cyanine dyes NIR1-NIR4 were converted to reactive N-succinyl estersusing diisopropylcarbodiimide (DIPCDI) and N-hydroxysuccinimide in thepresence of N-methylmorpholine in dimethylformamide (DMF) according tothe reaction scheme shown in FIG. 5A. A nearly quantitative yield(typically >98%) was observed using reversed phase HPLC, as shown inFIG. 5B. The formation of active ester was not only confirmed by reversephase HPLC, but also by reaction with benzylamine. FIG. 5B shows theHPLC of NIR2 (top chromatogram), as well as of its active ester (bottomchromatogram). Elution time for NIR2 and its active ester were 27.1 and29.0 min, respectively. When the active ester reacted with benzylamine,the resultant NIR2-benzylamine conjugate showed an elution time of 32.1min (HPLC profile not shown). The active ester was remarkably stable inwater. According to PLC analysis, less than 10% of the active ester washydrolyzed over a period of 20 days in water at 4° C.

In atypical experiment, 10 mg of dye, 30 μl of diisopropylcarbodiimide(DIPCDI; Aldrich), 50 μl of N-methylmorpholine (Aldrich), 22.0 mg ofN-hydroxysuccinimide (NHS; Aldrich), and 0.5 μl of dry dimethylformamide(DMF; Aldrich) were placed in a small round bottom flask under argonatmosphere. The mixture was stirred at room temperature for 3 hours. Themixture was then poured into ether, from which a solid precipitated.After centrifugation, the ether was decanted and the remaining solid waswashed four more times with ether and then dried in vacuo. According toHPLC analysis, more than 98% of the dye was converted to thecorresponding active ester.

The a-chloroacetamido-containing cyanine dyes NIR5-NIR8 can be convertedto the corresponding α-iodoacetamido-containing compounds. In general,the 60 -iodoacetamido functionality is a more reactive group forconjugation than the α-chloroacetamido functionality. Theiodoacetamido-containing cyanine dyes NIR9-12 were obtained from NIR4-8respectively via a halo-exchange reaction, using sodium iodide inrefluxing methanol by the synthetic method shown in FIG. 5C. Accordingto reversed-phase HPLC analysis, typically more than 98% of the chlorocompound was converted to the iodo compound. The elution times for thechloro and iodo compounds are e.g., 30.0 and 31.3 min for NIR6 and NIR10respectively.

In a typical experiment, 10 mg of 5-chloroacetamido-containing cyaninedye, 20 mg of sodium iodide, and 5 mL of methanol were placed in a smallround-bottomed flask under argon atmosphere. The mixture was heated toreflux for 2.5 hours. The solvent was evaporated to afford the5-iodoacetamido-containing cyanine dye. According to HPLC analysis, morethan 98% of chloro compound was converted to the iodo compound.

The coupling of haloacetamido-containing cyanine dyes with partnerscontaining a sulfhydryl group (—SH) was tested by the reaction of NIR10with a cysteine containing peptide (GRRGGGGYC) (SEQ IP) NO:17). HPLCtraces of NIR10 and the NIR10-peptide conjugate are shown in FIG. 5D.The elution time for the peptide conjugate was 28.8 min while that ofNIR10 was 31.3 min. The structure of the NIR10-peptide conjugate wasconfirmed by MALDI-TOF mass spectrometry. The fluorescence excitationand emission of the NIR10-peptide conjugate are shown in FIG. 5E. Thespectral properties of the NIR10-peptide conjugate were found to besimilar similar to those of the free cyanine dye (Ex=666 nm; Em=695 nm).These results indicated that iodoacetamido-containing cyanine dyes havea relatively high selectivity for sulfhydryl groups and could thereforebe useful for the specific labeling of sulfhydryl-containingbiomolecules, e.g., proteins, peptides.

In a typical procedure, the peptide, GRRGGGGYC (SEQ ID NO:17),synthesized by standard solid phase synthesis (3.0 mg), was dissolved in0.5 mL of 0.1 M aqueous NaHCO₃. To this solution was added 2.0 mg ofNIR10 dissolved in 0.5 mL of EtOH. The mixture was stirred at RTovernight. After removal of the solvent, the NIR10-peptide conjugate waspurified by reverse phase HPLC and analyzed by MALDI-TOF massspectrometry, M+1: expected=1545, found=1548.

Example 4 Imaging of NIR Dyes

An NIRF reflectance imaging system as described in Mahmood et al.,Radiology, 213:866-870 (1999) was used to image the new NIR dyes of theinvention and to compare them to ICG. Briefly, the system included alight-tight chamber equipped with a halogen white light source andexcitation bandpass filters, the first providing 610-650 nm excitationand 680-720 nm emission (“700 nm”), and the other 750-770 nm excitationand 800-820 nm emission (“800 nm”) (Omega Optical, Brattleboro, Vt.).

Equimolar NIR dyes and ICG were loaded into individual wells (0.16 nmolein 200 μl) in a clear bottom 96-well plate (Corning, Corning, N.Y.).Fluorescence was detected using a 12-bit monochrome CCD camera (Kodak,Rochester, N.Y.) equipped with a 12.5-75 mm zoom lens and emissionbandpass filters at 680-720 nm or 800-820 nm (Omega Optical,Brattleboro, Vt.). Exposure time was 10 sec per image. Images wereanalyzed using commercially available software (Kodak Digital Science 1Dsoftware, Rochester, N.Y.).

As shown in FIG. 6, the fluorescence signals of NIRs are well resolvedin this fluorescence imaging system. At 700 rum, only NIR2 and NIR4 weredetectable, while only NIR1 and NIR3 were detectable at 800 nm.Moreover, the NIR1 and NIR3 showed significantly better opticalproperties than ICG, as the signal intensities of NIR1 and NIR3 were 7-and 12-fold higher, respectively, than that of ICG.

Example 5 Synthesis of Enzyme-Sensitive Probe with Various Amounts ofNIR2

The enzyme-sensitive probes were synthesized by reacting partiallyPEGylated polylysine (0.1 mg, MW=500,000 Da) with various amounts ofNIR2 N-hydroxysuccinimide (NHS) ester, the concentrations of which were0.4, 2, 4, 8, 20, 40, and 80 μM in 20 mM NaHCO₃, at room temperature for3 hours. The NIR2-labeled polymers were then separated from excess lowmolecular weight reagents using a 50 kDa cutoff microconcentrator(Amicon, Beverly, Mass.). Based on NIR2 absorption measurement at 662nm, the average numbers of NIR2 fluorochrome per PGC were 0.2, 0.8, 1.4,2.4, 4.3, 5.7 and 7.0, respectively.

Example 6 Trypsin Activation of NIR2-PGC Probes

The activation of the NIRF probe was carried out in a 96-well plate withvarious NIR2-PGC probes. In each well, NIR2-PGC (40 pmole) in 200 μofphosphate-buffered saline (PBS) was incubated with 10 μL of trypsinsolution (0.05% trypsin, 0.53 mM EDTA, Mediatech, Herndon, Va.). Thereactions were monitored using a fluorescence microplate reader(Spectramax, Molecular Devices, Sunnyvale, Calif.) with excitation andemission wavelength at 662 and 684 nm, respectively. The reactions wererun in duplicate.

Example 7 Spectral Properties of Dyes

The absorption spectra of NIR1, NIR2, NIR3, and NIR4 are shown in FIG.4A. The difference in absorbance maxima between indodicarbocyanine dyeand indotricarbocyanine dye was about 100 nm. The terminal nucleuscontributes very little to the absorbance maxima compared to that of thebridging methine unit. The difference in absorbance maxima between 3/2heterocyclics (e.g., NIR2) compared to 2/2 homocycles (e.g., NIR4) wasonly 12 nm in water and 13 nm in ethanol. Excitation and emissionspectra of NIR1 and NIR2 are shown in FIG. 4B. Indodicarbocyanine dyesNIR3 and NIR4 had a 20 nm Stokes shift of the fluorescence emissionmaxima, while indotricarbocyanine dyes NIR1 and NIR2 exhibited a 30 nmStokes shift of the fluorescence maxima. The dyes had high molarextinction coefficients (ε) (i.e., above 250,000 L/mol cm¹). Quantumyields (QY) of the new fluorochromes varied from 0.23 to 0.43. Table 3summarizes the optical properties of the compounds. TABLE 3 OpticalProperties Stokes Com- λ_(max,abs) λ_(max,em) shift ε pound solvent (nm)(nm) (nm) (L mol⁻¹cm⁻¹⁾ QY NIR1 water 761 796 35 268,000 0.23 ethanol769 NIR2 water 662 684 22 250,000 0.34 ethanol 667 NIR3 water 750 777 27275,000 0.28 ethanol 756 NIR4 water 650 671 21 260,000 0.43 ethanol 654

Table 4, below, summarizes the optical properties of compounds NIR9-12.These compounds were stable, and exhibited relatively high molarextinction coefficients (200,000 to 250,000) and quantum yields (0.11 to0.24). TABLE 4 Optical Properties of the Synthesized Sulfhydryl-ReactiveFluorochromes Stokes λ_(max,abs) λ_(max,em) Shift ε compd solvent (nm)(nm) (nm) L mol⁻¹cm⁻¹ QY NIR10 H₂O/CH₃CN 666 695 29 218,000 0.24 (1:1)NIR9 H₂O/CH₃CN 763 803 40 224,000 0.11 (1:1) NIR12 H₂O/CH₃CN 667 697 30245,000 0.24 (2:1) NIR11 H₂O/CH₃CN 764 803 39 238,000 0.13 (2:1)

Example 8 Receptor-Targeted NIRF Probe

The synthetic strategy of the folate receptor-targeted probe is shown inFIG. 8. Folic acid was first synthesized as an activated ester byreacting it with N-hydroxysuccinimide (NHS) in dimethylformamide (DMF)using dicyclohexylcarbodiimide (DCC) as a condensing agent. One molarequivalent of 2,2′-(ethylenedioxy)bis-ethylamine (EDBEA) was thenattached to the activated folate ester; thereafter, NIR2 was coupled tothe newly generated amino group. Physical characterization indicatedthat the folate-NIR2 conjugate maintained all optical properties of freeNIR2.

Synthesis and purification of folate-EDBEA conjugate: Into around-bottomed flask were placed 477 mg (1 mmole) of folic aciddihydrate, 15 ml of anhydrous mixture was at heated at 50° C. for 5hours. After cooling the mixture to room temperature, 1 ml ofdiisopropylamine and 1.46 ml of EDBEA (Aldrich) were added. The mixturewas then stirred at room temperature for 24 hours. 20 ml of acetonitrilewas then added to the mixture to precipitate the product. The productwas washed three times with ethyl acetate, and then dried under vacuum.The crude product was purified using preparative HPLC (Rainin) using aC18-RP preparative column (flow rate=6 ml/minutes; eluant A, water with0.1% TFA; eluent B, 90% of acetonitrile and 10% of eluant A; starting at100% A for 5 minutes and then a linear gradient over 40 minutes to 60%A). The elution times for folate-EDBEA (alpha-link), and folate-EDBEA(gamma-linked) were 18.2 and 19.2 minutes, respectively. Massspectroscopic analysis provided a mass of 573 (calcd.=571).

Synthesis and purification of folate-EDBEA-NIR2 conjugate: A solution of3.8 mg of folate-EDBEA dissolved in 0.3 ml of 0.1 M aqueous NaHCO₃ wasadded to a solution of 6 mg of NIR2—CHS ester in 0.3 ml of DMF. Thereaction mixture was stirred at room temperature over night in the dark.The product was then precipitated by adding the mixture to acetone. Thecrude product was separated from the acetone and dried. Purification offolate-EDBEA-NIR2 was carried out using the same HPLC instrument asabove (flow rate=4 ml/min; eluant A, water with 0.1% TFA; eluent B, 90%of acetonitrile and 10% of eluant A; starting at 90% A for 5 minutes andthen a linear gradient over 40 minutes to 50% A). The elution time forfolate-EDBEA-NIR2 was 25.2 minutes. The successful conjugation of NIR2to the folate-EDBEA was confirmed by mass spectroscopic analysis, aswell as by fluorescent spectroscopy. Mass spectrum, calcd. 1401, found,1402. Fluorescence spectroscopy showed both the fluorescence emission ofNIR2 moiety (emission at 686 nm) and fluorescence emission of folatemoiety (emission at 430 nm).

Example 9 In vivo Imaging

The free NIR2 and the folate-NIR2 compounds were both tested in tumorbearing mice. These studies were conducted to a) determine thetolerability of the agents following intravenous (IV) injection, b)tumoral enhancement as a function of time and c) differential tumorenhancement of targeted vs. non-specific probe. The study utilizedfolate receptor (FR) positive OVCAR-5 tumors implanted into the mammaryfat pad of nude mice. All animals (n=5) tolerated the IV injection ofthe compounds without any signs of physiological changes over 2 weeks.Using the folate-derivatized NIR2, tumor enhancement became highlyapparent within a short time after IV injection and peaked at 4 hourspost-injection. A digitized photograph of one of the mice is shown inFIG. 9, illustrating that both large and small tumors can be easilydetected under the NIRF imaging system in vivo. When targeting andnontargeting compounds were compared in different subsets of animals,the folate receptor targeted compound resulted in much higher tumoralfluorescence when compared to the non-targeted probes. These resultsindicate that NIR2 is well tolerated and is receptor-targetable.

Example 10 Imaging of Cell Lines

The folate derivatized NIR2 was also evaluated in a human nasopharyngealepidermoid carcinoma, KB, cell line and a human fibrosarcoma, HT1080,cell line for its ability to improve the detection of FR positivecancers. These cell lines were selected because of putative FRoverexpression (KB) or lack of detectable FR expression (HT1080) (Ross,J. F., P. K. Chaudhuri, and M. Ratnam (1994) Differential regulation offolate receptor isoforms in normal and malignant tissues in vivo and inestablished cell lines. Physiologic and clinical implications. Cancer,73, 2432-43).

To confirm receptor expression levels, cellular binding/internalizationwas determined using ³H-folate. KB or HT1080 (10⁶ cells) grown in12-well plates were incubated at 37° C. for different times (1, 10, 30,60, or 120 minutes) with 50 nM ³H-folate (specific activity 34.5Ci/mmol, American Radiolabeled Chemical Inc, St. Louis, Mo.). At the endof the incubation, cells were harvested using 0.1% Triton X-100 and theradioactivity (pmol/10⁶ cells) was determined using a scintilltioncounter. For competitive inhibition studies, KB cells were incubatedwith different amounts of folic acid or NIR2-folate probe (5, 50, 500,and 5000 nM).

The HT1080 and KB cell lines were first characterized in terms of theirputative capability of ³H-folate binding and uptake. KB and HT1080 tumorcells were incubated with ³H-folate (50 nM) up to 120 minutes. Cellularbinding and uptake was quantified by scintillation counting. FIG. 10summarizes the cellular uptake and binding data and reveals significantuptake of ³H-folate by KB cells, but essentially no uptake by HT1080cells. For KB cells, 50% of saturation of available FR by ³H-folate wasreached in 20 min and uptake reached a plateau in 60 minutes. At peakmaximum 12 pmole of ³H-folate /10⁶ cells was observed under the chosenexperimental conditions. In competition assays, there was a 60% decreasein bound 3H-folate in the presence of an equimolar amount (50 nM) of thefree folic acid (4.97 pmole/10⁶ cells) or NIR2-folate probe (5.01pmole/10⁶ cells). As the concentration of the free folic acid orNIR2-folate probe was increased to 5000 nM, binding of ³H-folate alsodecreased to 15% of its initial value, free folic acid at 1.86 pmole/10⁶cells or NIR2-folate probe at 1.92 pmole/10⁶ cells. Competition by theNMR2-folate probe was similar to that of unconjugated folic acid. Theseresults confirmed that fluorochrome attachment does not interfere withFR binding.

Similar to previous uptake experiments, the NIR2-folate probe was testedin cell culture using KB and HT1080 cells grown at 70% confluency onglass cover slips. The culture medium was replaced with 0.5 mL of freshmedium containing 1 μM NIR2-folate probe and incubated for 1 hour at 37°C. Cells were washed three times and fluorescence microscopy wasperformed using an inverted epifluorescence microscope (Zeiss Axiovert,Thornwood, N.Y.).

To determine the localization of fluorescent folate within cells,fluorescence microscopy was performed on KB cells incubated with theNIR-2 folate probe. As shown in FIG. 11, the KB cells showed extensive,bright fluorescence signal whereas there was essentially no binding oruptake of the NIR2-folate probe in the negative control (HT1080 cells).Fluorescence signal was seen primarily in the distribution of the plasmamembrane of KB cells and in punctate vesicles in the interstitialcompartment.

Before testing the NIR2-folate probe in vivo, tumor expression of FR wasfurther characterized by immunohistology with FR recognizing Mab LK26.As shown in FIG. 12A, the staining showed strong immunoprecipitation inKB tumor tissues, indicating that the receptor remains overexpressedfollowing implantation. Antibody staining showed primarily membrane andcytoplasmic staining of the KB cells. In contrast, as shown in FIG. 12B,HT1080 tumor sections were essentially negative for folate receptor. Theresults of Hematoxylin-eosin staining are shown in FIG. 12C (KB cells)and 12D (HT1080 cells). Hematoxylin-eosin staining revealed multiplemitotic figures present in the rapidly proliferating HT1080fibrosarcoma, while relatively well differentiated epidermoid cells wereseen in the KB tumors.

Example 11 Imaging Solid Tumors In Vivo

To induce solid tumors, 10⁶ KB or HT1080 cells were injectedsubcutaneously into mammary fat pad and the lower abdomen of 30 nudemice (average weight 20 g). Within 7-17 days after implantations, eachmouse developed 3-4 tumors of 1-14 mm (mean 4.1 mm) in size. To studytumor heterogeneity, tumors with different sizes were included in theexperiments. For dual-tumor experiments, six mice were injected with 10⁶of KB and HT1080 cells on the ipsilateral and contralateral siderespectively.

Thirty-six mice bearing KB and/or HT1080 tumors (n=60 each) were dividedinto three groups so that each group had 12 mice collectively having atotal of 40 tumors; five mice collectively having a total of 18 KBtumors, five mice collectively having a total of 18 HT1080 tumors, andtwo mice with both KB and HT1080 tumors. Group 1 was injected with theNIR2-folate probe (2 nmole/mouse), group 2 received free NIR2fluorochrome (not conjugated to folate, 2 nmole/mouse), and group 3 wasinjected with the mixture of NIR2-folate probe (2 nmole/mouse) and freefolic acid (600 nmole folate/mouse). NIRF imaging was performed beforeand 1, 4, 24, 48 hours after tail vein injection of the probes. In twoanimals from each group, NIRF images were also acquired daily up to 7days (168 hours) to study the in vivo kinetics of the probe.

Following intravenous administration of the NIRF-folate probe, KB tumorsshowed significantly higher fluorescence signal intensity compared toHT1080 tumors. FIGS. 13A and 13D show the white light and NIRF imagesobtained 24 hours after intravenous injection of the NIR2-folate probein a representative animal. FIG. 13B is an enlarged image of the KB andHT1080 chest tumors. The former exhibits a relatively strongfluorescence signal, while the latter does not. FIG. 13C is an enlargedimage of the low abdomen tumor. The mice bearing KB tumors, tumoralfluorescence could be detected as early as 1 hour after administrationof the probe (728±109 AU), which peaked at 4 hours (1210 AU±127) andthen decreased (870 AU ±98 AU at 24 hours; 459 AU±48 AU at 48 hours and255 AU±39 at 72 hours).

FIG. 14 is a bar graph, which shows that in tumors of equal size, therewas a 2.4-fold (870 AU±98/366 AU±41, P<0.01) higher fluorescenceintensity in the FR-positive KB tumors compared with the control HT1080tumors at 24-hour images. In this set of experiments tumoral enhancementwas also compared with the free NIR2 dye. At the 24 hour time point,NIR-2 fluorochrome did not result in appreciably higher signal thanbackground. Similarly, in competition studies, fluorescence signal ofFR-positive KB tumor was reduced to that of FR-negative HT1080 tumors.The competition studies indicated that the availability of free folatewas able to compete off the receptor binding to NIR2-folate probe andthat fluorochrome-labelled ffolic acid can still be recognized by itsreceptor.

Tumor-to background contrast was measured and these ratios were plottedas a function of time after injection for the three experimental groups.The resulting graph is shown in FIG. 15. At the one hour time point, allagents had similar tumor/background ratios(y-axis) and these ratios wereonly moderately elevated in KB tumors. At 4 hours after injection, asignificantly higher tumor/background ratio for the NIR2-folate wasobserved when compared to the NIR2 compound. Importantly for clinicalapplications, tumor/background ratios remained elevated with this probefor at least 24-48 hours indicating its potential utility for endoscopicand intraoperative use (see FIG. 15). The tumoral fluorescence signalwas reduced rapidly after 72 hours (255 AU±39) and returned to thebaseline (115 AU±17) in 5 days. Organ distribution of the probe was alsoexamined after dissection. Highest fluorescence signal was observed inkidney because of high FR expression. Tumor, liver, lung and intestinewere at about a similar level.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An asymmetrical chromophore compound comprising the formula:

wherein L is a conjugated linker moiety; R-₁₋₁₂ are independentlyselected from the group consisting of hydrogen, substituted andunsubstituted alkyl groups, substituted and unsubstituted alkenylgroups, substituted and unsubstituted alkynyl groups, substituted andunsubstituted aryl groups, sulfur-containing functional groups,phosphorus-containing functional groups, oxygen-containing functionalgroups, and nitrogen-containing functional groups; and X and Y areindependently selected from the group consisting of oxygen, sulfur,nitrogen, and substituted or unsubstituted methylene.
 2. The compound ofclaim 1, wherein one or more of R₁₋₁₂ each independently comprises areactive group for conjugation to a macromolecule.
 3. The compound ofclaim 1, wherein one or more of R₁₋₁₂ comprise at least one substituentindependently selected from the group consisting of sulfate, sulfonate,phosphate, phosphonate, halide, nitro, nitrile, and carboxylate.
 4. Thecompound of claim 1, wherein L is (CH═CH—)CH.
 5. The compound of claim1, wherein L is (CH═CH—)₂CH.
 6. The compound of claim 1, wherein L is(CH═CH—)₃CH.
 7. The compound of claim 1, wherein L is (CH═CH—)₄CH. 8.The compound of claim 1, wherein L comprises one or more ringstructures.
 9. An asymmetrical chromophore compound comprising theformula:

wherein L is a conjugated linker moiety; R₇ and R₈ are independentlyselected from the group consisting of hydrogen, substituted andunsubstituted alkyl groups, substituted and unsubstituted alkenylgroups, substituted and unsubstituted alkynyl groups, substituted andunsubstituted aryl groups, sulfur-containing functional groups,phosphorus-containing functional groups, oxygen-containing functionalgroups, and nitrogen-containing functional groups; and; X and Y areindependently selected from the group consisting of oxygen, sulfur, andsubstituted or unsubstituted methylene; Z is a group of nonmetallicatoms necessary for forming a substituted or unsubstituted, condensedaromatic ring or ring system; R₁₃ is C(O)OR₁₄ or NHC(O)CH₂J; R₁₄ is H or

J is halo.
 10. The compound of claim 9, wherein R₁₄ is H.
 11. Thecompound of claim 9, wherein J is Cl or I.
 12. An asymmetricalchromophore compound comprising the formula:

wherein X is selected from the group consisting of:

wherein, n=2or3; R₈ is selected from the group consisting of hydrogen,substituted and unsubstituted alkyl groups, substituted andunsubstituted alkenyl groups, substituted and unsubstituted alkynylgroups, substituted and unsubstituted aryl groups, sulfur-containingfunctional groups, phosphorus-containing functional groups,oxygen-containing functional groups, and nitrogen-containing functionalgroups; R₁₃ is C(O)OR₁₄ or NHC(O)CH₂J; R₁₄ is H or

J is halo.
 13. The compound of claim 12, wherein R₈ is CH₃ or (CH₂)₄SO₃⁻.
 14. The compound of claim 12, wherein R₁₄ is H.
 15. The compound ofclaim 12, wherein J is Cl or I.
 16. An in vivo method of imaging atissue in a subject, the method comprising: a) conjugating to atargeting ligand a chromophore of claims 1 or 9; b) combining theconjugated chromophore with an excipient to form an administerableformulation; c) administering the formulation to the tissue; and d)detecting the conjugated chromophore in the tissue to provide afluorescence image of the tissue.
 17. The method of claim 16, whereinthe targeting ligand is a receptor binding ligand.
 18. The method ofclaim 16, wherein the tissue is a tumor tissue.
 19. The method of claim16, wherein the subject is mammal.
 20. The method of claim 16, whereinthe subject is a human.
 21. An in vitro method of imaging a tissue, themethod comprising: a) conjugating to a targeting ligand a chromophore ofclaims 1 or 9; b) contacting the conjugated chromophore with the tissue;and c) detecting the conjugated chromophore in the tissue to provide afluorescence image of the tissue.